Methods for Controlling Pests Using Rnai

ABSTRACT

The present invention relates to methods for controlling pest infestation using double stranded RNA molecules. The invention provides methods for producing transgenic cells expressing the double stranded RNA molecules, as well as compositions and commodity products containing or treated with such molecules.

FIELD OF THE INVENTION

The present invention relates generally to genetic control of pestinfestations. More specifically, the present invention relates torecombinant technologies for repressing or inhibiting expression oftarget coding sequences in a pest.

INTRODUCTION

Insect and other pests can cause injury and even death by their bites orstings. Additionally, many pests transmit bacteria and other pathogensthat cause diseases. For example, mosquitoes transmit pathogens thatcause malaria, yellow fever, encephalitis, and other diseases. Thebubonic plague, or black death, is caused by bacteria that infect ratsand other rodents. Compositions for controlling microscopic pestinfestations have been provided in the form of antibiotic, antiviral,and antifungal compositions. Methods for controlling infestations bypests, such as nematodes and insects, have typically been in the form ofchemical compositions that are applied to surfaces on which pestsreside, or administered to infested animals in the form of pellets,powders, tablets, pastes, or capsules.

Commercial crops are often the targets of insect attack. Substantialprogress has been made in the last a few decades towards developing moreefficient methods and compositions for controlling insect infestationsin plants. Chemical pesticides have been very effective in eradicatingpest infestations. However, there are several disadvantages to usingchemical pesticides. Not only are they potentially detrimental to theenvironment, but chemical pesticides are not selective and can pose harmto non-target flora and fauna. Chemical pesticides persist in theenvironment and generally are slow to be metabolized, if at all. Theyaccumulate in the food chain, and particularly in the higher predatorspecies. Accumulation of chemical pesticides results in the developmentof resistance to the agents and in species higher up the evolutionaryladder, they can act as mutagens and/or carcinogens and causeirreversible and deleterious genetic modifications.

Because of the dangers associated with chemical pesticides, biologicalapproaches have been developed for controlling pest infestations. Forexample, biological control using protein Cry3A from Bacillusthuringiensis have effectively controlled Colorado potato beetle larvaeeither as formulations sprayed onto the foliage or expressed in theleaves of potatoes. An alternative biological agent is double strandedRNA (dsRNA). Over the last few years, downregulation of genes (alsoreferred to as “gene silencing”) in multicellular organisms by means ofRNA interference has become a well-established technique.

RNA Interference (RNAi) provides a potentially powerful tool forcontrolling gene expression because of its specificity of targetselection and remarkably high efficiency in target mRNA suppression.RNAi refers to the process of sequence-specific post-transcriptionalgene silencing mediated by short interfering RNAs (siRNAs) (Zamore, P.et al., Cell 101:25-33 (2000); Fire, A. et al., Nature 391:806 (1998);Hamilton et al., Science 286, 950-951 (1999); Lin et al., Nature402:128-129 (1999)). While the mechanics underlying RNAi are not fullycharacterized, it is thought that the presence of dsRNA in cellstriggers RNAi by activating the ribonuclease III enzyme Dicer (Zamore,P. et al., (2000); Hammond et al., Nature 404, 293 (2000)). Dicerprocesses the dsRNA into short pieces called short interfering RNAs(siRNAs), which are about 21 to about 23 nucleotides long and compriseabout 19 base pair duplexes (Zamore et al., (2000); Elbashir et al.,Genes Dev., 15, 188 (2001)). Following delivery into cells, the siRNAmolecules associate with an endonuclease complex, commonly referred toas an RNA-induced silencing complex (RISC), which brings together theantisense strand of the siRNA and the cellular mRNA gene target. RISCcleaves the mRNA, which is then released and degraded. Importantly, RISCis then capable of degrading additional copies of the target mRNA.

Accordingly, the present invention provides methods and compositions forcontrolling pest infestation by repressing, delaying, or otherwisereducing gene expression within a particular pest.

SUMMARY OF THE INVENTION

In one aspect, the invention provides an isolated polynucleotidesequence comprising a nucleic acid sequence set forth in SEQ ID NOs: 1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 49-158, 159, 160, 163, 168, 173,178, 183, 188, 193, 198, 203, 208, 215, 220, 225, 230, 247, 249, 251,253, 255, 257, 259, 275-472, 473, 478, 483, 488, 493, 498, 503, 513,515, 517, 519, 521, 533-575, 576, 581, 586, 591, 596, 601, 603, 605,607, 609, 621-767, 768, 773, 778, 783, 788, 793, 795, 797, 799, 801,813-862, 863, 868, 873, 878, 883, 888, 890, 892, 894, 896, 908-1040,1041, 1046, 1051, 1056, 1061, 1071, 1073, 1075, 1077, 1079, 1081, 1083,1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099, 1101, 1103, 1105, 1107,1109, 1111, 1113, 1161-1571, 1572, 1577, 1582, 1587, 1592, 1597, 1602,1607, 1612, 1617, 1622, 1627, 1632, 1637, 1642, 1647, 1652, 1657, 1662,1667, 1672, 1677, 1682, 1684, 1686, 1688, 1690, 1692, 1694, 1696, 1698,1700, 1702, 1704, 1730-2039, 2040, 2045, 2050, 2055, 2060, 2065, 2070,2075, 2080, 2085, 2090, 2095, 2100, 2102, 2104, 2106, 2108, 2120-2338,2339, 2344, 2349, 2354, 2359, 2364, 2366, 2368, 2370, 2372, 2384-2460,2461, 2466, 2471, 2476 and 2481. In one embodiment, a double strandedribonucleotide sequence is produced from the expression of apolynucleotide sequence, wherein contact of said ribonucleotide sequenceby a pest inhibits the growth of said pest. In a further embodiment,contact of the sequence inhibits expression of a nucleotide sequencesubstantially complementary to said sequence. In another embodiment, acell is transformed with the polynucleotide. In a further embodiment,the cell is a bacterial, yeast, or algal cell. In a still furtherembodiment, a food product, such as stored grains, pet food, or powderedchocolate, comprises the cell transformed with the polynucleotide. Inyet another embodiment, a composition, such as a spray, powder, pellet,gel, capsule, food product, garment bag, and book, comprising thepolynucleotide. In yet another embodiment, the invention provides apesticide comprising the polynucleotide. In another embodiment, theinvention provides a method for protecting an object, such as wood,tree, book binding, cloth, and a food storage container, from pestinfestation, comprising treating said surface with a compositioncomprising the polynucleotide.

In another aspect, the invention provides a polynucleotide sequencehaving at least 70% sequence identity to a nucleic acid sequence setforth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 49-158,159, 160, 163, 168, 173, 178, 183, 188, 193, 198, 203, 208, 215, 220,225, 230, 247, 249, 251, 253, 255, 257, 259, 275-472, 473, 478, 483,488, 493, 498, 503, 513, 515, 517, 519, 521, 533-575, 576, 581, 586,591, 596, 601, 603, 605, 607, 609, 621-767, 768, 773, 778, 783, 788,793, 795, 797, 799, 801, 813-862, 863, 868, 873, 878, 883, 888, 890,892, 894, 896, 908-1040, 1041, 1046, 1051, 1056, 1061, 1071, 1073, 1075,1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099,1101, 1103, 1105, 1107, 1109, 1111, 1113, 1161-1571, 1572, 1577, 1582,1587, 1592, 1597, 1602, 1607, 1612, 1617, 1622, 1627, 1632, 1637, 1642,1647, 1652, 1657, 1662, 1667, 1672, 1677, 1682, 1684, 1686, 1688, 1690,1692, 1694, 1696, 1698, 1700, 1702, 1704, 1730-2039, 2040, 2045, 2050,2055, 2060, 2065, 2070, 2075, 2080, 2085, 2090, 2095, 2100, 2102, 2104,2106, 2108, 2120-2338, 2339, 2344, 2349, 2354, 2359, 2364, 2366, 2368,2370, 2372, 2384-2460, 2461, 2466, 2471, 2476 and 2481. In oneembodiment, a double stranded ribonucleotide sequence is produced fromthe expression of a polynucleotide sequence, wherein contact of saidribonucleotide sequence by a pest inhibits the growth of said pest. In afurther embodiment, contact of the sequence inhibits expression of anucleotide sequence substantially complementary to said sequence. Inanother embodiment, a cell is transformed with the polynucleotide. In afurther embodiment, the cell is a bacterial, yeast, or algal cell. In astill further embodiment, a food product, such as stored grains, petfood, or powdered chocolate, comprises the cell transformed with thepolynucleotide. In yet another embodiment, a composition, such as aspray, powder, pellet, gel, capsule, food product, garment bag, andbook, comprising the polynucleotide. In yet another embodiment, theinvention provides a pesticide comprising the polynucleotide. In anotherembodiment, the invention provides a method for protecting an object,such as wood, tree, book binding, cloth, and a food storage container,from pest infestation, comprising treating said surface with acomposition comprising the polynucleotide.

In another aspect, the invention provides a method for controlling pestinfestation, comprising exposing a pest to a composition comprising apolynucleotide sequence that inhibits a pest biological activity. In oneembodiment, the polynucleotide sequence is set forth in any of SEQ IDNOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 49-158, 159, 160, 163,168, 173, 178, 183, 188, 193, 198, 203, 208, 215, 220, 225, 230, 247,249, 251, 253, 255, 257, 259, 275-472, 473, 478, 483, 488, 493, 498,503, 513, 515, 517, 519, 521, 533-575, 576, 581, 586, 591, 596, 601,603, 605, 607, 609, 621-767, 768, 773, 778, 783, 788, 793, 795, 797,799, 801, 813-862, 863, 868, 873, 878, 883, 888, 890, 892, 894, 896,908-1040, 1041, 1046, 1051, 1056, 1061, 1071, 1073, 1075, 1077, 1079,1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099, 1101, 1103,1105, 1107, 1109, 1111, 1113, 1161-1571, 1572, 1577, 1582, 1587, 1592,1597, 1602, 1607, 1612, 1617, 1622, 1627, 1632, 1637, 1642, 1647, 1652,1657, 1662, 1667, 1672, 1677, 1682, 1684, 1686, 1688, 1690, 1692, 1694,1696, 1698, 1700, 1702, 1704, 1730-2039, 2040, 2045, 2050, 2055, 2060,2065, 2070, 2075, 2080, 2085, 2090, 2095, 2100, 2102, 2104, 2106, 2108,2120-2338, 2339, 2344, 2349, 2354, 2359, 2364, 2366, 2368, 2370, 2372,2384-2460, 2461, 2466, 2471, 2476 and 2481.

In other embodiments, the invention provides for the use of the isolatednucleotide sequence, the double stranded ribonucleotide sequence, thecell, the composition, or the pesticide for preventing or treating aninfestation, such as insect, nematode, or fungal infestation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1-LD: Survival of L. decemlineata on artificial diet treated withdsRNA. Insects of the second larval stage were fed diet treated with 50μl of topically-applied solution of dsRNA (targets or gfp control). Dietwas replaced with fresh diet containing topically-applied dsRNA after 7days. The number of surviving insects were assessed at days 2, 5, 7, 8,9, & 13. The percentage of surviving larvae was calculated relative today 0 (start of assay). Target LD006: (SEQ ID NO: 178); Target LD007(SEQ ID NO: 183); Target LD010 (SEQ ID NO: 188); Target LD011 (SEQ IDNO: 193); Target LD014 (SEQ ID NO: 198); gfp dsRNA (SEQ ID NO: 235).

FIG. 2-LD: Survival of L. decemlineata on artificial diet treated withdsRNA. Insects of the second larval stage were fed diet treated with 50μl of topically-applied solution of dsRNA (targets or gfp control). Dietwas replaced with fresh diet only after 7 days. The number of survivinginsects was assessed at days 2, 5, 6, 7, 8, 9, 12, & 14. The percentageof surviving larvae was calculated relative to day 0 (start of assay).Target LD001 (SEQ ID NO: 163); Target LD002 (SEQ ID NO: 168); TargetLD003 (SEQ ID NO: 173); Target LD015 (SEQ ID NO: 215); Target LD016 (SEQID NO: 220); gfp dsRNA (SEQ ID NO: 235).

FIG. 3-LD: Average weight of L. decemlineata larvae on potato leaf discstreated with dsRNA. Insects of the second larval stage were fed leafdiscs treated with 20 μl of a topically-applied solution (10 ng/μl) ofdsRNA (target LD002 or gfp). After two days the insects were transferredon to untreated leaves every day.

FIG. 4-LD: Survival of L. decemlineata on artificial diet treated withshorter versions of target LD014 dsRNA and concatemer dsRNA. Insects ofthe second larval stage were fed diet treated with 50 μl oftopically-applied solution of dsRNA (gfp or targets). The number ofsurviving insects were assessed at days 3, 4, 5, 6, & 7. The percentageof surviving larvae were calculated relative to day 0 (start of assay).

FIG. 5-LD: Survival of L. decemlineata larvae on artificial diet treatedwith different concentrations of dsRNA of target LD002 (a), target LD007(b), target LD010 (c), target LD011 (d), target LD014 (e), target LD015(f), LD016 (g) and target LD027 (h). Insects of the second larval stagewere fed diet treated with 50 μl of topically-applied solution of dsRNA.Diet was replaced with fresh diet containing topically-applied dsRNAafter 7 days. The number of surviving insects were assessed at regularintervals. The percentage of surviving larvae were calculated relativeto day 0 (start of assay).

FIG. 6-LD. Effects of E. coli strains expressing dsRNA target LD010 onsurvival of larvae of the Colorado potato beetle, Leptinotarsadecemlineata, over time. The two bacterial strains were tested inseparate artificial diet-based bioassays: (a) AB309-105; data points forpGBNJ003 and pGN29 represent average mortality values from 5 differentbacterial clones, (b) BL21DE3); data points for pGBNJ003 and pGN29represent average mortality values from 5 different and one singlebacterial clones, respectively. Error bars represent standarddeviations.

FIG. 7-LD. Effects of different clones of E. coli strains (a) AB309-105and (b) BL21(DE3) expressing dsRNA target LD010 on survival of larvae ofthe Colorado potato beetle, Leptinotarsa decemlineata, 12 days postinfestation. Data points are average mortality values for each clone forpGN29 and pGBNJ003. Clone 1 of AB309-105 harbouring plasmid pGBNJ003showed 100% mortality towards CPB at this timepoint, Error barsrepresent standard deviations.

FIG. 8-LD. Effects of different clones of E. coli strains (a) AB309-105and (b) BL21(DE3) expressing dsRNA target LD010 on growth anddevelopment of larval survivors of the Colorado potato beetle,Leptinotarsa decemlineata, 7 days post infestation. Data points are %average larval weight values for each clone (one clone for pGN29 andfive clones for pGBNJ003) based on the data of Table 10. Diet onlytreatment represents 100% normal larval weight.

FIG. 9-LD. Survival of larvae of the Colorado potato beetle,Leptinotarsa decemlineata, on potato plants sprayed by double-strandedRNA-producing bacteria 7 days post infestation. Number of larvalsurvivors were counted and expressed in terms of % mortality. Thebacterial host strain used was the RNaseIII-deficient strain AB309-105.Insect gene target was LD010.

FIG. 10-LD. Growth/developmental delay of larval survivors of theColorado potato beetle, Leptinotarsa decemlineata, fed on potato plantssprayed with dsRNA-producing bacteria 11 days post infestation. Thebacterial host strain used was the RNaseIII-deficient strain AB309-105.Data figures represented as percentage of normal larval weight; 100% ofnormal larval weight given for diet only treatment. Insect gene targetwas LD010. Error bars represent standard deviations.

FIG. 11-LD. Resistance to potato damage caused by larvae of the Coloradopotato beetle, Leptinotarsa decemlineata, by double-strandedRNA-producing bacteria 7 days post infestation. Left, plant sprayed with7 units of bacteria AB309-105 containing the pGN29 plasmid; right, plantsprayed with 7 units of bacteria Ab309-105 containing the pGBNJO03plasmid. One unit is defined as the equivalent of 1 ml of a bacterialsuspension at OD value of 1 at 600 nm. Insect gene target was LD010.

FIG. 12-LD. Survival of L. decemlineata adults on potato leaf discstreated with dsRNA. Young adult insects were feddouble-stranded-RNA-treated leaf discs for the first two days and werethen placed on untreated potato foliage. The number of surviving insectswere assessed regularly; mobile insects were recorded as insects whichwere alive and appeared to move normally; moribund insects were recordedas insects which were alive but appeared sick and slow moving—theseinsects were not able to right themselves once placed on their backs.Target LD002 (SEQ ID NO: 168); Target LD010 (SEQ ID NO: 188); TargetLD014 (SEQ ID NO: 198); Target LD016 (SEQ ID NO: 220); gfp dsRNA (SEQ IDNO: 235).

FIG. 13-LD. Effects of bacterial produced target double-stranded RNAagainst larvae of L. decemlineata. Fifty μl of an OD 1 suspension ofheat-treated bacteria expressing dsRNA (SEQ ID NO: 188) was appliedtopically onto the solid artificial diet in each well of a 48-wellplate. CPB larvae at L2 stage were placed in each well. At day 7, apicture was taken of the CPB larvae in a plate containing (a) diet withbacteria expressing target 10 double-stranded RNA, (b) diet withbacteria harbouring the empty vector pGN29, and, (c) diet only.

FIG. 14-LD Effects on CPB larval survival and growth of differentamounts of inactivated E. coli AB309-105 strain harbouring plasmidpGBNJ003 topically applied to potato foliage prior to insectinfestation. Ten L1 larvae were fed treated potato for 7 days. Amount ofbacterial suspension sprayed on plants: 0.25 U, 0.08 U, 0.025 U, 0.008 Uof target 10 and 0.25 U of pGN29 (negative control; also included isMilli-Q water). One unit (U) is defined as the equivalent bacterialamount present in 1 ml of culture with an optical density value of 1 at600 nm. A total volume of 1.6 ml was sprayed on to each plant. Insectgene target was LD010.

FIG. 15-LD Resistance to potato damage caused by CPB larvae byinactivated E. coli AB309-105 strain harbouring plasmid pGBNJO03 sevendays post infestation. (a) water, (b) 0.25 U E. coli AB309-105harbouring pGN29, (c) 0.025 U E. coli AB309-105 harbouring pGBNJ003, (d)0.008 U E. coli AB309-105 harbouring pGBNJ003. One unit (U) is definedas the equivalent bacterial amount present in 1 ml of culture with anoptical density value of 1 at 600 nm. A total volume of 1.6 ml wassprayed on to each plant. Insect gene target was LD010.

FIG. 1-PC: Effects of ingested target dsRNAs on survival and growth ofP. cochleariae larvae. Neonate larvae were fed oilseed rape leaf discstreated with 25 μl of topically-applied solution of 0.1 μg/μl dsRNA(targets or gfp control). After 2 days, the insects were transferredonto fresh dsRNA-treated leaf discs. At day 4, larvae from one replicatefor every treatment were collected and placed in a Petri dish containingfresh untreated oilseed rape foliage. The insects were assessed at days2, 4, 7, 9 & 11. (a) Survival of E. varivestis larvae on oilseed rapeleaf discs treated with dsRNA. The percentage of surviving larvae wascalculated relative to day 0 (start of assay). (b) Average weights of P.cochleariae larvae on oilseed rape leaf discs treated with dsRNA.Insects from each replicate were weighed together and the average weightper larva determined. Error bars represent standard deviations. Target1: SEQ ID NO: 473; target 3: SEQ ID NO: 478; target 5: SEQ ID NO: 483-;target 10: SEQ ID NO: 488; target 14: SEQ ID NO: 493; target 16: SEQ IDNO: 498; target 27: SEQ ID NO: 503; gfp dsRNA: SEQ ID NO: 235.

FIG. 2-PC: Survival of P. cochleariae on oilseed rape leaf discs treatedwith different concentrations of dsRNA of (a) target PC010 and (b)target PC027. Neonate larvae were placed on leaf discs treated with 25μl of topically-applied solution of dsRNA. Insects were transferred tofresh treated leaf discs at day 2. At day 4 for target PC010 and day 5for target PC027, the insects were transferred to untreated leaves. Thenumber of surviving insects were assessed at days 2, 4, 7, 8, 9 & 11 forPC010 and 2, 5, 8, 9 & 12 for PC027. The percentage of surviving larvaewas calculated relative to day 0 (start of assay).

FIG. 3-PC: Effects of E. coli strain AB309-105 expressing dsRNA targetPC010 on survival of larvae of the mustard leaf beetle, P. cochleariae,over time. Data points for each treatment represent average mortalityvalues from 3 different replicates. Error bars represent standarddeviations. Target 10: SEQ ID NO: 488

FIG. 1-EV: Survival of E. varivestis larvae on bean leaf discs treatedwith dsRNA. Neonate larvae were fed bean leaf discs treated with 25 μlof topically-applied solution of 1 μg/μl dsRNA (targets or gfp control).After 2 days, the insects were transferred onto fresh dsRNA-treated leafdiscs. At day 4, larvae from one treatment were collected and placed ina plastic box containing fresh untreated bean foliage. The insects wereassessed for mortality at days 2, 4, 6, 8 & 10. The percentage ofsurviving larvae was calculated relative to day 0 (start of assay).Target 5: SEQ ID NO: 576; target 10: SEQ ID NO: 586; target 15: SEQ IDNO: 591; target 16: SEQ ID NO: 596; gfp dsRNA: SEQ ID NO: 235.

FIG. 2-EV: Effects of ingested target dsRNAs on survival of E.varivestis adults and resistance to snap bean foliar insect damage. (a)Survival of E. varivestis adults on bean leaf treated with dsRNA. Adultswere fed bean leaf discs treated with 75 μl of topically-appliedsolution of 0.1 μg/μl dsRNA (targets or gfp control). After 24 hours,the insects were transferred onto fresh dsRNA-treated leaf discs. Aftera further 24 hours, adults from one treatment were collected and placedin a plastic box containing potted fresh untreated whole bean plants.The insects were assessed for mortality at days 4, 5, 6, 7, 8, & 11. Thepercentage of surviving adults was calculated relative to day 0 (startof assay). Target 10: SEQ ID NO: 586; target 15: SEQ ID NO: 591; target16: SEQ ID NO: 596; gfp dsRNA: SEQ ID NO: 235; (b) Resistance to beanfoliar damage caused by adults of the E. varivestis by dsRNA. Wholeplants containing insects from one treatment (see (a)) were checkedvisually for foliar damage on day 9. (i) target 10; (ii) target 15;(iii) target 16; (iv) gfp dsRNA; (v) untreated.

FIG. 1-TC: Survival of T. castaneum larvae on artificial diet treatedwith dsRNA of target 14. Neonate larvae were fed diet based on aflour/milk mix with 1 mg dsRNA target 14. Control was water (withoutdsRNA) in diet. Four replicates of 10 first instar larvae per replicatewere performed for each treatment. The insects were assessed forsurvival as average percentage means at days 6, 17, 31, 45 and 60. Thepercentage of surviving larvae was calculated relative to day 0 (startof assay). Error bars represent standard deviations. Target TC014: SEQID NO: 878.

FIG. 1-MP: Effect of ingested target 27 dsRNA on the survival of Myzuspersicae nymphs. First instars were placed in feeding chamberscontaining 50 μl of liquid diet with 2 μg/μl dsRNA (target 27 or gfpdsRNA control). Per treatment, 5 feeding chambers were set up with 10instars in each feeding chamber. Number of survivors were assessed at 8days post start of bioassay. Error bars represent standard deviations.Target MP027: SEQ ID NO: 1061; gfp dsRNA: SEQ ID NO: 235.

FIG. 1-NL: Survival of Nilaparvata lugens on liquid artificial diettreated with dsRNA. Nymphs of the first to second larval stage were feddiet supplemented with 2 mg/ml solution of dsRNA targets in separatebioassays: (a) NL002, NL003, NL005, NL010; (b) NL009, NL016; (c) NL014,NL018; (d) NL013, NL015, NL021. Insect survival on targets were comparedto diet only and diet with gfp dsRNA control at same concentration. Dietwas replaced with fresh diet containing dsRNA every two days. The numberof surviving insects were assessed every day

FIG. 2-NL: Survival of Nilaparvata lugens on liquid artificial diettreated with different concentrations of target dsRNA NL002. Nymphs ofthe first to second larval stage were fed diet supplemented with 1, 0.2,0.08, and 0.04 mg/ml (final concentration) of NL002. Diet was replacedwith fresh diet containing dsRNA every two days. The numbers ofsurviving insects were assessed every day.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a means for controlling pest infestationsby exposing a pest to a target coding sequence thatpost-transcriptionally represses or inhibits a requisite biologicalfunction in the pest. Following exposure to a target sequence, thetarget forms a the dsRNA corresponding to part or whole of an essentialpest gene and causes down regulation of the pest target via RNAinterference (RNAi). As a result of the down regulation of mRNA, thedsRNA prevents expression of the target pest protein and hence causesdeath, growth arrest, or sterility of the pest.

The present invention finds application in any area where it isdesirable to inhibit viability, growth, development or reproduction of apest, or to decrease pathogenicity or infectivity of a pest. Practicalapplications include, but are not limited to, (1) protecting plantsagainst pest infestation; (2) pharmaceutical or veterinary use in humansand animals (for example to control, treat, or prevent insect infectionsin humans and animals); (3) protecting materials against damage causedby pests; and (4) protecting perishable materials (such as foodstuffs,seed, etc.) against damage caused by pests.

Administering or exposing a double stranded ribonucleic acid molecule toa pest results in one or more of the following attributes: reduction infeeding by the pest, reduction in viability of the pest, death of thepest, inhibition of differentiation and development of the pest, absenceof or reduced capacity for sexual reproduction by the pest, muscleformation, juvenile hormone formation, juvenile hormone regulation, ionregulation and transport, maintenance of cell membrane potential, aminoacid biosynthesis, amino acid degradation, sperm formation, pheromonesynthesis, pheromone sensing, antennae formation, wing formation, legformation, development and differentiation, egg formation, larvalmaturation, digestive enzyme formation, haemolymph synthesis, haemolymphmaintenance, neurotransmission, cell division, energy metabolism,respiration, apoptosis, and any component of a eukaryotic cells'cytoskeletal structure, such as, for example, actins and tubulins. Anyone or any combination of these attributes can result in an effectiveinhibition of pest infestation.

All technical terms employed in this specification are commonly used inbiochemistry, molecular biology and agriculture; hence, they areunderstood by those skilled in the field to which this inventionbelongs. Those technical terms can be found, for example in: MOLECULARCLONING: A LABORATORY MANUAL, 3rd ed., vol. 1-3, ed. Sambrook andRussel, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,2001; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, ed. Ausubel et al., GreenePublishing Associates and Wiley-Interscience, New York, 1988 (withperiodic updates); SHORT PROTOCOLS IN MOLECULAR BIOLOGY: A COMPENDIUM OFMETHODS FROM CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, 5^(th) ed., vol.1-2, ed. Ausubel et al., John Wiley & Sons, Inc., 2002; GENOME ANALYSIS:A LABORATORY MANUAL, vol. 1-2, ed. Green et al., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1997.

Various techniques using PCR are described, for example, in Innis etal., PCR PROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS, Academic Press,San Diego, 1990 and in Dieffenbach and Dveksler, PCR PRIMER: ALABORATORY MANUAL, 2^(nd) ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 2003. PCR-primer pairs can be derived from knownsequences by known techniques such as using computer programs intendedfor that purpose, e.g., Primer, Version 0.5, 1991, Whitehead Institutefor Biomedical Research, Cambridge, Mass. Methods for chemical synthesisof nucleic acids are discussed, for example, in Beaucage & Caruthers,Tetra. Letts. 22: 1859-62 (1981), and Matteucci & Caruthers, J. Am.Chem. Soc. 103: 3185 (1981).

Restriction enzyme digestions, phosphorylations, ligations, andtransformations were done as described in Sambrook et al., MOLECULARCLONING: A LABORATORY MANUAL, 2nd ed. (1989), Cold Spring HarborLaboratory Press. All reagents and materials used for the growth andmaintenance of bacterial cells were obtained from Aldrich Chemicals(Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), Invitrogen(Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.) unlessotherwise specified.

Biological activity refers to the biological behavior and effects of aprotein or peptide and its manifestations on a pest. For example, aninventive RNAi may prevent translation of a particular mRNA, therebyinhibiting the biological activity of the protein encoded by the mRNA orother biological activity of the pest.

In the present description, an RNAi molecule may inhibit a biologicalactivity in a pest, resulting in one or more of the followingattributes: reduction in feeding by the pest, reduction in viability ofthe pest, death of the pest, inhibition of differentiation anddevelopment of the pest, absence of or reduced capacity for sexualreproduction by the pest, muscle formation, juvenile hormone formation,juvenile hormone regulation, ion regulation and transport, maintenanceof cell membrane potential, amino acid biosynthesis, amino aciddegradation, sperm formation, pheromone synthesis, pheromone sensing,antennae formation, wing formation, leg formation, development anddifferentiation, egg formation, larval maturation, digestive enzymeformation, haemolymph synthesis, haemolymph maintenance,neurotransmission, cell division, energy metabolism, respiration,apoptosis, and any component of a eukaryotic cells' cytoskeletalstructure, such as, for example, actins and tubulins.

Complementary DNA (cDNA) refers to single-stranded DNA synthesized froma mature mRNA template. Though there are several methods, cDNA is mostoften synthesized from mature (fully spliced) mRNA using the enzymereverse transcriptase. This enzyme operates on a single strand of mRNA,generating its complementary DNA based on the pairing of RNA base pairs(A, U, G, C) to their DNA complements (T, A, C, G). Two nucleic acidstrands are substantially complementary when at least 85% of their basespair.

Desired Polynucleotide: a desired polynucleotide of the presentinvention is a genetic element, such as a promoter, enhancer, orterminator, or gene or polynucleotide that is to be transcribed and/ortranslated in a transformed cell that comprises the desiredpolynucleotide in its genome. If the desired polynucleotide comprises asequence encoding a protein product, the coding region may be operablylinked to regulatory elements, such as to a promoter and a terminator,that bring about expression of an associated messenger RNA transcriptand/or a protein product encoded by the desired polynucleotide. Thus, a“desired polynucleotide” may comprise a gene that is operably linked inthe 5′- to 3′-orientation, a promoter, a gene that encodes a protein,and a terminator. Alternatively, the desired polynucleotide may comprisea gene or fragment thereof, in a “sense” or “antisense” orientation, thetranscription of which produces nucleic acids that may affect expressionof an endogenous gene in the host cell. A desired polynucleotide mayalso yield upon transcription a double-stranded RNA product upon thatinitiates RNA interference of a gene to which the desired polynucleotideis associated. A desired polynucleotide of the present invention may bepositioned within a vector, such that the left and right bordersequences flank or are on either side of the desired polynucleotide. Thepresent invention envisions the stable integration of one or moredesired polynucleotides into the genome of at least one host cell. Adesired polynucleotide may be mutated or a variant of its wild-typesequence. It is understood that all or part of the desiredpolynucleotide can be integrated into the genome of a host. It also isunderstood that the term “desired polynucleotide” encompasses one ormore of such polynucleotides. Thus, a vector of the present inventionmay comprise one, two, three, four, five, six, seven, eight, nine, ten,or more desired polynucleotides.

“Exposing” encompasses any method by which a pest may come into contactwith a dsRNA, wherein the dsRNA comprises annealed complementarystrands, one of which has a nucleotide sequence which is complementaryto at least part of the nucleotide sequence of a pest target gene to bedown-regulated. A pest may be exposed to the dsRNA by direct uptake(e.g. by feeding), which does not require expression of dsRNA within thepest. Alternatively, a pest may come into direct contact with acomposition comprising the dsRNA. For example, a pest may come intocontact with a surface or material treated with a composition comprisinga dsRNA. A dsRNA may be expressed by a prokaryotic (for instance, butnot limited to, a bacterial) or eukaryotic (for instance, but notlimited to, a yeast) host cell or host organism.

Foreign: “foreign,” with respect to a nucleic acid, means that thatnucleic acid is derived from non-host organisms. According to thepresent invention, foreign DNA or RNA represents nucleic acids that arenaturally occurring in the genetic makeup of viruses, mammals, fish orbirds, but are not naturally occurring in the host that is to betransformed. Thus, a foreign nucleic acid is one that encodes, forinstance, a polypeptide that is not naturally produced by thetransformed host. A foreign nucleic acid does not have to encode aprotein product.

Gene: refers to a polynucleotide sequence that comprises control andcoding sequences necessary for the production of a polypeptide orprecursor. The polypeptide can be encoded by a full length codingsequence or by any portion of the coding sequence. A gene may constitutean uninterrupted coding sequence or it may include one or more introns,bound by the appropriate splice junctions. Moreover, a gene may containone or more modifications in either the coding or the untranslatedregions that could affect the biological activity or the chemicalstructure of the expression product, the rate of expression, or themanner of expression control. Such modifications include, but are notlimited to, mutations, insertions, deletions, and substitutions of oneor more nucleotides. In this regard, such modified genes may be referredto as “variants” of the “native” gene.

Genetic element: a “genetic element” is any discreet nucleotide sequencesuch as, but not limited to, a promoter, gene, terminator, intron,enhancer, spacer, 5′-untranslated region, 3′-untranslated region, orrecombinase recognition site.

Genetic modification: stable introduction of a nucleic acid into thegenome of certain organisms by applying methods in molecular and cellbiology.

“Gene suppression” or “down-regulation of gene expression” or“inhibition of gene expression” are used interchangeably and refer to ameasurable or observable reduction in gene expression or a completeabolition of detectable gene expression, at the level of protein productand/or mRNA product from the target gene. Down-regulation or inhibitionof gene expression is “specific” when down-regulation or inhibition ofthe target gene occurs without manifest effects on other genes of thepest.

Depending on the nature of the target gene, down-regulation orinhibition of gene expression in cells of a pest can be confirmed byphenotypic analysis of the cell or the whole pest or by measurement ofmRNA or protein expression using molecular techniques such as RNAsolution hybridization, nuclease protection, Northern hybridization,reverse transcription, gene expression monitoring with a microarray,antibody binding, enzyme-linked immunosorbent assay (ELISA), Westernblotting, radioimmunoassay (RIA), other immunoassays, orfluorescence-activated cell analysis (FACS).

Gymnosperm, as used herein, refers to a seed plant that bears seedwithout ovaries. Examples of gymnosperms include conifers, cycads,ginkgos, and ephedras.

Homology, as used herein relates to sequences; Protein, or nucleotidesequences are likely to be homologous if they show a “significant” levelof sequence similarity or more preferably sequence identity. Trulyhomologous sequences are related by divergence from a common ancestorgene, Sequence homologs can be of two types: (i) where homologs exist indifferent species they are known as orthologs. e.g. the α-globin genesin mouse and human are orthologs; (ii) paralogues are homologous genesin within a single species. e.g. the α- and β-globin genes in mouse areparalogs.

Host cell: refers to a microorganism, a prokaryotic cell, a eukaryoticcell, or cell line cultured as a unicellular entity that may be, or hasbeen, used as a recipient for a recombinant vector or other transfer ofpolynucleotides, and includes the progeny of the original cell that hasbeen transfected. The progeny of a single cell may not necessarily becompletely identical in morphology or in genomic or total DNA complementas the original parent due to natural, accidental, or deliberatemutation.

Introduction: as used herein, refers to the insertion of a nucleic acidsequence into a cell, by methods including infection, transfection,transformation, or transduction.

Insect pests as used herein pests are include but are not limited to:from the order Lepidoptera, for example, Acleris spp., Adoxophyes spp.,Aegeria spp., Agrotis spp., Alabama argillaceae, Amylois spp.,Anticarsia gemmatalis, Archips spp, Argyrotaenia spp., Autographa spp.,Busseola fusca, Cadra cautella, Carposina nipponensis, Chilo spp.,Choristoneura spp., Clysia ambiguella, Cnaphalocrocis spp., Cnephasiaspp., Cochylis spp., Coleophora spp., Crocidolomia binotalis,Cryptophlebia leucotreta, Cydia spp., Diatraea spp., Diparopsiscastanea, Earias spp., Ephestia spp., Eucosma spp., Eupoeciliaambiguella, Euproctis spp., Euxoa spp., Grapholita spp., Hedyanubiferana, Heliothis spp., Hellula undalis, Hyphantria cunea, Keiferialycopersicella, Leucoptera scitella, Lithocollethis spp., Lobesiabotrana, Lymantria spp., Lyonetia spp., Malacosoma spp., Mamestrabrassicae, Manduca sexta, Operophtera spp., Ostrinia Nubilalis, Pammenespp., Pandemis spp., Panolis flammea, Pectinophora gossypiella,Phthorimaea operculella, Pieris rapae, Pieris spp., Plutella xylostella,Prays spp., Scirpophaga spp., Sesamia spp., Sparganothis spp.,Spodoptera spp., Synanthedon spp., Thaumetopoea spp., Tortrix spp.,Trichoplusia ni and Yponomeuta spp.;

from the order Coleoptera, for example, Agriotes spp., Anthonomus spp.,Atomaria linearis, Chaetocnema tibialis, Cosmopolites spp., Curculiospp., Dermestes spp., Epilachna spp., Eremnus spp., Leptinotarsadecemlineata, Lissorhoptrus spp., Melolontha spp., Orycaephilus spp.,Otiorhynchus spp., Phlyctinus spp., Popillia spp., Psylliodes spp.,Rhizopertha spp., Scarabeidae, Sitophilus spp., Sitotroga spp., Tenebriospp., Tribolium spp. and Trogoderma spp.;

from the order Orthoptera, for example, Blatta spp., Blattella spp.,Gryllotalpa spp., Leucophaea maderae, Locusta spp., Periplaneta ssp.,and Schistocerca spp.;

from the order Isoptera, for example, Reticulitemes spp.;

from the order Psocoptera, for example liposcelis ssp.;

from the order Anoplura, for example Haematopinus spp., Linognathusspp., Pediculus spp., Pemphigus spp. and Phylloxera spp.;

from the order Mallophaga, for example, Damalinea spp. and Trichodectesspp.;

from the order Thysanoptera, for example, Franklinella spp.,Hercinothrips spp., Taeniothrips spp., Thrips palmi, Thrips tabaci andScirtothrips aurantii;

from the order Heteroptera, for example, Cimex spp., Distantiellatheobroma, Dysdercus spp., Euchistus spp., Eurygaster spp., Leptocorisaspp., Nezara spp., Piesma spp., Rhodnius spp., Sahlbergella singularis,Scotinophara spp., Triatoma spp., Miridae family spp. such as Lygushesperus and Lygus lineoloris, Lygaeidae family spp. such as Blissusleucopterus, and Pentatomidae family spp.;

from the order Homoptera, for example, Aleurothrixus floccosus,Aleyrodes brassicae, Aonidiella spp., Aphididae, Aphis spp., Aspidiotusspp., Bemisia tabaci, Ceroplaster spp., Chrysomphalus aonidium,Chrysomphalus dictyospermi, Coccus hesperidum, Empoasca spp., Eriosomalarigerum, Erythroneura spp., Gascardia spp., Laodelphax spp., Lacaniumcorni, Lepidosaphes spp., Macrosiphus spp., Myzus spp., Nehotettix spp.,Nilaparvata spp., Paratoria ssp., Pemphigus spp., Planococcus spp.,Pseudaulacaspis spp., Pseudococcus spp., Psylla sp., Pulvinariaaethiopica, Quadraspidiotus spp., Rhopalosiphum spp., Saissetia spp.,Scaphoideus spp., Schizaphis spp., Sitobion spp., Trialeurodesvaporariorum, Trioza erytreae and Unaspis citri;

from the order Hymenoptera, for example, Acromyrmex, Atta spp., Cephusspp., Diprion spp., Diprionidae, Gilpinia polytoma, Hoplocampa spp.,Lasius spp., Monomorium pharaonis, Neodiprion spp., Solenopsis spp. andVespa ssp.;

from the order Diptera, for example, Aedes spp., Antherigona soccata,Bibio hortulanus, Calliphora erythrocephala, Ceratitis spp., Chrysomyiaspp., Culex spp., Cuterebra spp., Dacus spp., Drosophila melanogaster,Fannia spp., Gastrophilus spp., Glossina spp., Hypoderma spp.,Hyppobosca spp., Liriomysa spp., Lucilia spp., Melanagromyza spp., Muscassp., Oestrus spp., Orseolia spp., Oscinella frit, Pegomyia hyoscyami,Phorbia spp., Rhagoletis pomonella, Sciara spp., Stomoxys spp., Tabanusspp., Tannia spp. and Tipula spp.,

from the order Siphonaptera, for example, Ceratophyllus spp. undXenopsylla cheopis and

from the order Thysanura, for example Lepisma saccharina.

Monocotyledonous plant (monocot) is a flowering plant having embryoswith one cotyledon or seed leaf, parallel leaf veins, and flower partsin multiples of three. Examples of monocots include, but are not limitedto turfgrass, maize, rice, oat, wheat, barley, sorghum, orchid, iris,lily, onion, and palm.

Pest or target pest refers to insects, arachnids, crustaceans, fungi,bacteria, viruses, nematodes, flatworms, roundworms, pinworms,hookworms, tapeworms, trypanosomes, schistosomes, botflies, fleas,ticks, mites, and lice and the like that are pervasive in the humanenvironment. A pest may ingest or contact one or more cells, tissues, orproducts produced by an organism transformed with a double stranded genesuppression agent, as well as a material or surface treated with adouble stranded gene suppression agent.

Nematodes, or roundworms, are one of the most common phyla of animals,with over 20,000 different described species (over 15,000 areparasitic). They are ubiquitous in freshwater, marine, and terrestrialenvironments, where they often outnumber other animals in bothindividual and species counts, and are found in locations as diverse asAntarctica and oceanic trenches. Further, there are a great manyparasitic forms, including pathogens in most plants and animals.

Nematode pests of a particular interest include, for example, A.caninum, A. ceylancium, H. contortus, O. ostertagi, C. elegans, C.briggsae, P. pacificus, S. stercoralis, S. ratti, P. trichosuri, M.arenaria, M. chitwoodi, M. hapla, M. incognita, M. javanica, M.paraensis, G. rostochiensis, G. pallida, H. glycines, H. schattii, P.penetrans, P. vulnus, R. similis, Z. punctata, A. suum, T. canis, B.malayi, D. immitis, O. volvulus, T. vulpis, T. spiralis, X. index. A.duodenale, A. lumbricoides, as well as species from the followinggenera: Aphelenchoides, Nacobbus, Ditylenchus, Longidorus, Trichodorus,and Bursaphelenchus.

Normal cell refers to a cell of an untransformed phenotype or exhibitinga morphology of a non-transformed cell of the tissue type beingexamined.

Operably linked: combining two or more molecules in such a fashion thatin combination they function properly in a cell. For instance, apromoter is operably linked to a structural gene when the promotercontrols transcription of the structural gene.

Orthologs are genes that are related by vertical descent from a commonancestor and encode proteins with the same function in differentspecies. Due to their separation following a speciation event, orthologsmay diverge, but usually have similarity at the sequence and structurelevels. Two genes that are derived from a common ancestor and encodeproteins with similar function are referred to as orthologous.Identification of orthologs is critical for reliable predictions of genefunction in newly sequenced genomes.

“Pest control agent”, or “gene suppression agent” refers to a particularRNA molecule comprising a first RNA segment and a second RNA segment,wherein the complementarity between the first and the second RNAsegments results in the ability of the two segments to hybridize in vivoand in vitro to form a double stranded molecule. It may generally bepreferable to include a third RNA segment linking and stabilizing thefirst and second sequences such that a stem can be formed linkedtogether at one end of each of the first and second segments by thethird segment to forms a loop, so that the entire structure forms into astem and loop structure, or even more tightly hybridizing structures mayform into a stem-loop knotted structure. Alternatively, a symmetricalhairpin could be formed without a third segment in which there is nodesigned loop, but for steric reasons a hairpin would create its ownloop when the stem is long enough to stabilize itself. The first and thesecond RNA segments will generally lie within the length of the RNAmolecule and be substantially inverted repeats of each other and linkedtogether by the third RNA segment. The first and the second segmentscorrespond invariably and not respectively to a sense and an antisensesequence with respect to the target RNA transcribed from the target genein the target insect pest that is suppressed by the ingestion of thedsRNA molecule.

The pest control agent can also be a substantially purified (orisolated) nucleic acid molecule and more specifically nucleic acidmolecules or nucleic acid fragment molecules thereof from a genomic DNA(gDNA) or cDNA library. Alternatively, the fragments may comprisesmaller oligonucleotides having from about 15 to about 250 nucleotideresidues, and more preferably, about 15 to about 30 nucleotide residues.

Pesticide refers to any substance or mixture of substances intended forpreventing, destroying, repelling, or mitigating any pest. A pesticidemay be a chemical substance or biological agent used against pestsincluding insects, pathogens, weeds, nematodes, and microbes thatcompete with humans for food, destroy property, spread disease, or are anuisance.

Phenotype is a distinguishing feature or characteristic of an organism,which may be altered according to the present invention by integratingone or more “desired polynucleotides” and/or screenable/selectablemarkers into the genome of at least one cell of a transformed organism.The “desired polynucleotide(s)” and/or markers may confer a change inthe phenotype of a transformed organism, by modifying any one of anumber of genetic, molecular, biochemical, physiological, ormorphological characteristics or properties of the transformed cell ororganism as a whole.

Plant and plant tissue: a “plant” is any of various photosynthetic,eukaryotic, multicellular organisms of the kingdom Plantaecharacteristically producing embryos, containing chloroplasts, andhaving cellulose cell walls. A part of a plant, i.e., a “plant tissue”may be treated according to the methods of the present invention toprevent pest infestation on the plant or on the part of the plant. Manysuitable plant tissues can be treated according to the present inventionand include, but are not limited to, somatic embryos, pollen, leaves,stems, calli, stolons, microtubers, and shoots. Thus, the presentinvention envisions the treatment of angiosperm and gymnosperm plantssuch as acacia, alfalfa, apple, apricot, artichoke, ash tree, asparagus,avocado, banana, barley, beans, beet, birch, beech, blackberry,blueberry, broccoli, brussels sprouts, cabbage, canola, cantaloupe,carrot, cassaya, cauliflower, cedar, a cereal, celery, chestnut, cherry,Chinese cabbage, citrus, clementine, clover, coffee, corn, cotton,cowpea, cucumber, cypress, eggplant, elm, endive, eucalyptus, fennel,figes, fir, geranium, grape, grapefruit, groundnuts, ground cherry, gumhemlock, hickory, kale, kiwifruit, kohlrabi, larch, lettuce, leek,lemon, lime, locust, pine, maidenhair, maize, mango, maple, melon,millet, mushroom, mustard, nuts, oak, oats, okra, onion, orange, anornamental plant or flower or tree, papaya, palm, parsley, parsnip, pea,peach, peanut, pear, peat, pepper, persimmon, pigeon pea, pine,pineapple, plantain, plum, pomegranate, potato, pumpkin, radicchio,radish, rapeseed, raspberry, rice, rye, sorghum, sallow, soybean,spinach, spruce, squash, strawberry, sugarbeet, sugarcane, sunflower,sweet potato, sweet corn, tangerine, tea, tobacco, tomato, trees,triticale, turf grasses, turnips, a vine, walnut, watercress,watermelon, wheat, yarns, yew, and zucchini.

According to the present invention “plant tissue” also encompasses plantcells. Plant cells include suspension cultures, callus, embryos,meristematic regions, callus tissue, leaves, roots, shoots,gametophytes, sporophytes, pollen, seeds and microspores. Plant tissuesmay be at various stages of maturity and may be grown in liquid or solidculture, or in soil or suitable media in pots, greenhouses or fields. Aplant tissue also refers to any clone of such a plant, seed, progeny,propagule whether generated sexually or asexually, and descendents ofany of these, such as cuttings or seed.

Promoter is intended to mean a nucleic acid, preferably DNA that bindsRNA polymerase and/or other transcription regulatory elements. As withany promoter, the promoters of the current invention will facilitate orcontrol the transcription of DNA or RNA to generate an mRNA moleculefrom a nucleic acid molecule that is operably linked to the promoter. Asstated earlier, the RNA generated may code for a protein or polypeptideor may code for an RNA interfering, or antisense molecule.

Polynucleotide is a nucleotide sequence, comprising a gene codingsequence or a fragment thereof, a promoter, an intron, an enhancerregion, a polyadenylation site, a translation initiation site, 5′ or 3′untranslated regions, a reporter gene, a selectable marker or the like.The polynucleotide may comprise single stranded or double stranded DNAor RNA. The polynucleotide may comprise modified bases or a modifiedbackbone. The polynucleotide may be genomic, an RNA transcript (such asan mRNA) or a processed nucleotide sequence (such as a cDNA). Thepolynucleotide may comprise a sequence in either sense or antisenseorientations.

An isolated polynucleotide is a polynucleotide sequence that is not inits native state, e.g., the polynucleotide is comprised of a nucleotidesequence not found in nature or the polynucleotide is separated fromnucleotide sequences with which it typically is in proximity or is nextto nucleotide sequences with which it typically is not in proximity.

Recombinant nucleotide sequence refers to a nucleic acid molecule thatcontains a genetically engineered modification through manipulation viamutagenesis, restriction enzymes, and the like.

RNA interference (RNAi) refers to sequence-specific or gene-specificsuppression of gene expression (protein synthesis) that is mediated byshort interfering RNA (siRNA).

Sequence identity: as used herein, “sequence identity” or “identity” inthe context of two nucleic acid sequences includes reference to theresidues in the two sequences which are the same when aligned formaximum correspondence over a specified region.

As used herein, percentage of sequence identity means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

“Sequence identity” has an art-recognized meaning and can be calculatedusing published techniques. See COMPUTATIONAL MOLECULAR BIOLOGY, Lesk,ed. (Oxford University Press, 1988), BIOCOMPUTING: INFORMATICS ANDGENOME PROJECTS, Smith, ed. (Academic Press, 1993), COMPUTER ANALYSIS OFSEQUENCE DATA, PART I, Griffin & Griffin, eds., (Humana Press, 1994),SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, Von Heinje ed., Academic Press(1987), SEQUENCE ANALYSIS PRIMER, Gribskov & Devereux, eds. (MacmillanStockton Press, 1991), and Carillo & Lipton, SIAM J. Applied Math. 48:1073 (1988). Methods commonly employed to determine identity orsimilarity between two sequences include but are not limited to thosedisclosed in GUIDE TO HUGE COMPUTERS, Bishop, ed., (Academic Press,1994) and Carillo & Lipton, supra. Methods to determine identity andsimilarity are codified in computer programs. Preferred computer programmethods to determine identity and similarity between two sequencesinclude but are not limited to the GCG program package (Devereux et al.,Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschulet al., J. Mol. Biol. 215: 403 (1990)), and FASTDB (Brutlag et al.,Comp. App. Biosci. 6: 237 (1990)).

Short hairpin RNA (shRNA) are short single-stranded RNAs having a highdegree of secondary structure such that a portion of the RNA strandforms a hairpin loop.

Short interfering RNA (siRNA) refers to double-stranded RNA moleculesfrom about 10 to about 30 nucleotides long that are named for theirability to specifically interfere with gene protein expression.

Target sequence refers to a nucleotide sequence in a pest that isselected for suppression or inhibition by double stranded RNAtechnology. A target sequence encodes an essential feature or biologicalactivity within a pest.

Transcriptional terminators: The expression DNA constructs of thepresent invention typically have a transcriptional termination region atthe opposite end from the transcription initiation regulatory region.The transcriptional termination region may be selected, for stability ofthe mRNA to enhance expression and/or for the addition ofpolyadenylation tails added to the gene transcription product.Translation of a nascent polypeptide undergoes termination when any ofthe three chain-termination codons enters the A site on the ribosome.Translation termination codons are UAA, UAG, and UGA.

Transformation: A process by which a nucleic acid is stably insertedinto the genome of an organism. Transformation may occur under naturalor artificial conditions using various methods well known in the art.Transformation may rely on any known method for the insertion of nucleicacid sequences into a prokaryotic or eukaryotic host cell, includingmicroorganism-mediated transformation, viral infection, whiskers,electroporation, microinjection, polyethylene glycol-treatment, heatshock, lipofection, and particle bombardment.

Transgenic organism comprises at least one cell in which an exogenousnucleic acid has been stably integrated. A transgenic organism accordingto the invention is for instance a bacterial, or eukaryotic, such as ayeast, host cell or host organism. The bacterium can be chosen from thegroup comprising Gram-negative and Gram-positive bacteria, such as, butnot limited to, Escherichia spp. (e.g. E. coli), Bacillus spp. (e.g. B.thuringiensis), Rhizobium spp., Lactobacilllus spp., Lactococcus spp.,etc. The yeast can be chosen from the group comprising Saccharomycesspp., etc.

Variant: a “variant,” as used herein, is understood to mean a nucleotidesequence that deviates from the standard, or given, nucleotide or aminoacid sequence of a particular gene or protein. The terms, “isoform,”“isotype,” and “analog” also refer to “variant” forms of a nucleotidesequence. “Variant” may also refer to a “shuffled gene” such as thosedescribed in Maxygen-assigned patents.

It is understood that the present invention is not limited to theparticular methodology, protocols, vectors, and reagents, etc.,described herein, as these may vary. It is also to be understood thatthe terminology used herein is used for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention. It must be noted that as used herein and in theappended claims, the singular forms “a,” “an,” and “the” include pluralreference unless the context clearly dictates otherwise. Thus, forexample, a reference to “a gene” is a reference to one or more genes andincludes equivalents thereof known to those skilled in the art and soforth.

I. Target Pests

The present invention provides methodology and constructs forcontrolling pest infestations by administering, or otherwise exposing,to a pest a target coding sequence that post-transcriptionally repressesor inhibits a requisite biological function in the pest. As used herein,the term “pest” refers to insects, arachnids, crustaceans, fungi,bacteria, viruses, nematodes, flatworms, roundworms, pinworms,hookworms, tapeworms, trypanosomes, schistosomes, botflies, fleas,ticks, mites, and lice and the like that are pervasive in the humanenvironment. A pest may ingest or contact one or more cells, tissues, orproducts produced by an organism transformed with a double stranded genesuppression agent, as well as a surface or material treated with adouble stranded gene suppression agent.

A “pest resistance” trait is a characteristic of a transgenic host thatcauses the host to be resistant to attack from a pest that typicallyinflicts damage to the host. Such pest resistance can arise from anatural mutation or more typically from incorporation of recombinant DNAthat confers pest resistance. To impart pest resistance to a transgenichost, a recombinant DNA can, for example, be transcribed into a RNAmolecule that forms a dsRNA molecule within the tissues or fluids of therecombinant host. The dsRNA molecule is comprised in part of a segmentof RNA that is identical to a corresponding RNA segment encoded from aDNA sequence within a pest that prefers to feed on the recombinant host.Expression of the gene within the target pest is suppressed by thedsRNA, and the suppression of expression of the gene in the target pestresults in the host being pest resistant.

Suitable pests include any organism that causes damage to anotherorganism. The invention contemplates insect, nematode, and fungal pestsin particular.

Insect as used herein can be any insect, meaning any organism belongingto the Kingdom Animals, more specific to the Phylum Arthropoda, and tothe Class Insecta or the Class Arachnida. The methods of the inventionare applicable to all insects and that are susceptible to gene silencingby RNA interference and that are capable of internalisingdouble-stranded RNA from their immediate environment.

In one embodiment of the invention, the insect may belong to thefollowing orders: Acari, Araneae, Anoplura, Coleoptera, Collembola,Dermaptera, Dictyoptera, Diplura, Diptera, Embioptera, Ephemeroptera,Grylloblatodea, Hemiptera, Homoptera, Hymenoptera, Isoptera,Lepidoptera, Mallophaga, Mecoptera, Neuroptera, Odonata, Orthoptera,Phasmida, Plecoptera, Protura, Psocoptera, Siphonaptera, Siphunculata,Thysanura, Strepsiptera, Thysanoptera, Trichoptera, and Zoraptera.

In preferred, but non-limiting, embodiments and methods of the inventionthe insect is chosen from the group consisting of:

(1) an insect which is a plant pest, such as but not limited toNilaparvata spp. (e.g. N. lugens (brown planthopper)); Laodelphax spp.(e.g. L. striatellus (small brown planthopper)); Nephotettix spp. (e.g.N. virescens or N. cincticeps (green leafhopper), or N. nigropictus(rice leafhopper)); Sogatella spp. (e.g. S. furcifera (white-backedplanthopper)); Blissus spp. (e.g. B. leucopterus leucopterus (chinchbug)); Scotinophora spp. (e.g. S. vermidulate (rice blackbug));Acrosternum spp. (e.g. A. hilare (green stink bug)); Parnara spp. (e.g.P. guttata (rice skipper)); Chilo spp. (e.g. C. suppressalis (ricestriped stem borer), C. auricilius (gold-fringed stem borer), or C.polychrysus (dark-headed stem borer)); Chilotraea spp. (e.g. C.polychrysa (rice stalk borer)); Sesamia spp. (e.g. S. inferens (pinkrice borer)); Tryporyza spp. (e.g. T. innotata (white rice borer), or T.incertulas (yellow rice borer)); Cnaphalocrocis spp. (e.g. C. medinalis(rice leafroller)); Agromyza spp. (e.g. A. oryzae (leafminer), or A.parvicornis (corn blot leafminer)); Diatraea spp. (e.g. D. saccharalis(sugarcane borer), or D. grandiosella (southwestern corn borer));Narnaga spp. (e.g. N. aenescens (green rice caterpillar)); Xanthodesspp. (e.g. X. transversa (green caterpillar)); Spodoptera spp. (e.g. S.frugiperda (fall armyworm), S. exigua (beet armyworm), S. littoralis(climbing cutworm) or S. praefica (western yellowstriped armyworm));Mythimna spp. (e.g. Mythmna (Pseudaletia) seperata (armyworm));Helicoverpa spp. (e.g. H. zea (corn earworm)); Colaspis spp. (e.g. C.brunnea (grape colaspis)); Lissorhoptrus spp. (e.g. L. oryzophilus (ricewater weevil)); Echinocnemus spp. (e.g. E. squamos (rice plant weevil));Diclodispa spp. (e.g. D. armigera (rice hispa)); Oulema spp. (e.g. O.oryzae (leaf beetle); Sitophilus spp. (e.g. S. oryzae (rice weevil));Pachydiplosis spp. (e.g. P. oryzae (rice gall midge)); Hydrellia spp.(e.g. H. griseola (small rice leafminer), or H. sasakii (rice stemmaggot)); Chlorops spp. (e.g. C. oryzae (stem maggot)); Ostrinia spp.(e.g. O. nubilalis (European corn borer)); Agrotis spp. (e.g. A. ipsilon(black cutworm)); Elasmopalpus spp. (e.g. E. Iignosellus (lessercornstalk borer)); Melanotus spp. (wireworms); Cyclocephala spp. (e.g.C. borealis (northern masked chafer), or C. immaculata (southern maskedchafer)); Popillia spp. (e.g. P. japonica (Japanese beetle));Chaetocnema spp. (e.g. C. pulicaria (corn flea beetle)); Sphenophorusspp. (e.g. S. maidis (maize billbug)); Rhopalosiphum spp. (e.g. R.maidis (corn leaf aphid)); Anuraphis spp. (e.g. A. maidiradicis (cornroot aphid)); Melanoplus spp. (e.g. M. femurrubrum (redleggedgrasshopper) M. differentialis (differential grasshopper) or M.sanguinipes (migratory grasshopper)); Hylemya spp. (e.g. H. platura(seedcorn maggot)); Anaphothrips spp. (e.g. A. obscrurus (grassthrips)); Solenopsis spp. (e.g. S. milesta (thief ant)); or spp. (e.g.T. urticae (twospotted spider mite), T. cinnabarinus (carmine spidermite); Helicoverpa spp. (e.g. H. zea (cotton bollworm), or H. armigera(American bollworm)); Pectinophora spp. (e.g. P. gossypiella (pinkbollworm)); Earias spp. (e.g. E. vittella (spotted bollworm)); Heliothisspp. (e.g. H. virescens (tobacco budworm)); Anthonomus spp. (e.g. A.grandis (boll weevil)); Pseudatomoscelis spp. (e.g. P. seriatus (cottonfleahopper)); Trialeurodes spp. (e.g. T. abutiloneus (banded-wingedwhitefly) T. vaporariorum (greenhouse whitefly)); Bemisia spp. (e.g. B.argentifolii (silverleaf whitefly)); Aphis spp. (e.g. A. gossypii(cotton aphid), A. mellifera); Lygus spp. (e.g. L. lineolaris (tarnishedplant bug) or L. hesperus (western tarnished plant bug)); Euschistusspp. (e.g. E. conspersus (consperse stink bug)); Chlorochroa spp. (e.g.C. sayi (Say stinkbug)); Nezara spp. (e.g. N. viridula (southern greenstinkbug)); Thrips spp. (e.g. T. tabaci (onion thrips)); Frankliniellaspp. (e.g. F. fusca (tobacco thrips), or F. occidentalis (western flowerthrips)); Leptinotarsa spp. (e.g. L. decemlineata (Colorado potatobeetle), L. juncta (false potato beetle), or L. texana (Texan falsepotato beetle)); Lema spp. (e.g. L. trilineata (three-lined potatobeetle)); Epitrix spp. (e.g. E. cucumeris (potato flea beetle), E.hirtipennis (flea beetle), or E. tuberis (tuber flea beetle)); Epicautaspp. (e.g. E. vittata (striped blister beetle)); Empoasca spp. (e.g. E.fabae (potato leafhopper)); Myzus spp. (e.g. M. persicae (green peachaphid)); Paratrioza spp. (e.g. P. cockerelli (psyllid)); Conoderus spp.(e.g. C. falli (southern potato wireworm), or C. vespertinus (tobaccowireworm)); Phthorimaea spp. (e.g. P. operculella (potato tuberworm));Macrosiphum spp. (e.g. M. euphorbiae (potato aphid)); Thyanta spp. (e.g.T. pallidovirens (redshouldered stinkbug)); Phthorimaea spp. (e.g. P.operculella (potato tuberworm)); Helicoverpa spp. (e.g. H. zea (tomatofruitworm); Keiferia spp. (e.g. K lycopersicella (tomato pinworm));Limonius spp. (wireworms); Manduca spp. (e.g. M sexta (tobaccohornworm), or M. quinquemaculata (tomato hornworm)); Liriomyza spp.(e.g. L. sativae, L. trifolli or L. huidobrensis (leafminer));Drosophila spp. (e.g. D. melanogaster, D. yakuba, D. pseudoobscura or D.simulans); Carabus spp. (e.g. C. granulatus); Chironomus spp. (e.g. C.tentanus); Ctenocephalides spp. (e.g. C. felis (cat flea)); Diaprepesspp. (e.g. D. abbreviatus (root weevil)); Ips spp. (e.g. L. pini (pineengraver)); Tribolium spp. (e.g. T. castaneum (red floor beetle));Glossina spp. (e.g. G. morsitans (tsetse fly)); Anopheles spp. (e.g. A.gambiae (malaria mosquito)); Helicoverpa spp. (e.g. H. armigera (AfricanBollworm)); Acyrthosiphon spp. (e.g. A. pisum (pea aphid)); Apis spp.(e.g. A. melifera (honey bee)); Homalodisca spp. (e.g. H. coagulate(glassy-winged sharpshooter)); Aedes spp. (e.g. Ae. aegypti (yellowfever mosquito)); Bombyx spp. (e.g. B. mori (silkworm), B. mandarina);Locusta spp. (e.g. L. migratoria (migratory locust)); Boophilus spp.(e.g. B. microplus (cattle tick)); Acanthoscurria spp. (e.g. A.gomesiana (red-haired chololate bird eater)); Diploptera spp. (e.g. D.punctata (pacific beetle cockroach)); Heliconius spp. (e.g. H. erato(red passion flower butterfly) or H. melpomene (postman butterfly));Curculio spp. (e.g. C. glandium (acorn weevil)); Plutella spp. (e.g. P.xylostella (diamondback moth)); Amblyomma spp. (e.g. A. variegatum(cattle tick)); Anteraea spp. (e.g. A. yamamai (silkmoth)); Belgica spp.(e.g. B. antartica), Bemisa spp. (e.g. B. tabaci), Bicyclus spp.,Biphillus spp., Callosobruchus spp., Choristoneura spp., Cicindela spp.,Culex spp., Culicoides spp., Diaphorina spp., Diaprepes spp., Euclidiaspp., Glossina spp., Gryllus spp., Hydropsyche spp., Julodis spp.,Lonomia spp., Lutzomyia spp., Lysiphebus spp, Meladema spp, Mycetophagusspp., Nasonia spp., Oncometopia spp., Papilio spp., Pediculus spp.,Plodia spp., Rhynchosciara spp., Sphaerius spp., Toxoptera spp.,Trichoplusa spp., and Armigeres spp. (e.g. A. subalbatus);

(2) an insect capable of infesting or injuring humans and/or animalssuch as, but not limited to those with piercing-sucking mouthparts, asfound in Hemiptera and some Hymenoptera and Diptera such as mosquitoes,bees, wasps, lice, fleas and ants, as well as members of the Arachnidaesuch as ticks and mitesorder, class or family of Acarina (ticks andmites) e.g. representatives of the families Argasidae, Dermanyssidae,Ixodidae, Psoroptidae or Sarcoptidae and representatives of the speciesAmblyomma spp., Anocentor spp., Argas spp., Boophilus spp., Cheyletiellaspp., Chorioptes spp., Demodex spp., Dermacentor spp., Dermanyssus spp.,Haemophysalis spp., Hyalomma spp., Ixodes spp., Lynxacarus spp.,Mesostigmata spp., Notoedres spp., Ornithodoros spp., Ornithonyssusspp., Otobius spp., otodectes spp., Pneumonyssus spp., Psoroptes spp.,Rhipicephalus spp., Sarcoptes spp., or Trombicula spp.; Anoplura(sucking and biting lice) e.g. representatives of the species Bovicolaspp., Haematopinus spp., Linognathus spp., Menopon spp., Pediculus spp.,Pemphigus spp., Phylloxera spp., or Solenopotes spp.; Diptera (flies)e.g. representatives of the species Aedes spp., Anopheles spp.,Calliphora spp., Chrysomyia spp., Chrysops spp., Cochliomyia spp., Culexspp., Culicoides spp., Cuterebra spp., Dermatobia spp., Gastrophilusspp., Glossina spp., Haematobia spp., Haematopota spp., Hippobosca spp.,Hypoderma spp., Lucilia spp., Lyperosia spp., Melophagus spp., Oestrusspp., Phaenicia spp., Phlebotomus spp., Phornia spp., Sarcophaga spp.,Simulium spp., Stomoxys spp., Tabanus spp., Tannia spp. or Tipula spp.;Mallophaga (biting lice) e.g. representatives of the species Damalinaspp., Felicola spp., Heterodoxus spp. or Trichodectes spp.; orSiphonaptera (wingless insects) e.g. representatives of the speciesCeratophyllus spp., spp., Pulex spp., or Xenopsylla spp; Cimicidae (truebugs) e.g. representatives of the species Cimex spp., Tritominae spp.,Rhodinius spp., or Triatoma spp.

and

(3) an insect that causes unwanted damage to substrates or materials,such as insects that attack foodstuffs, seeds, wood, paint, plastic,clothing etc.

The methods of the invention are applicable to all nematodes and thatare susceptible to gene silencing by RNA interference and that arecapable of internalising double-stranded RNA from their immediateenvironment.

In one embodiment of the invention, the nematode may belong to thefamily of the Heteroderidae, encompassing the genera Heterodera andGlobodera.

In preferred, but non-limiting, embodiments and methods of the inventionthe insect is chosen from the group comprising but not limited to:

(1) a nematode which is a plant pathogenic nematode, such as but notlimited to: Meloidogyne spp. (e.g. M. incognita, M javanica, M.graminicola, M. arenaria, M. chitwoodi, M hapla or M. paranaensis);Heterodera spp. (e.g. H. oryzae, H. glycines, H. zeae or H. schachtii);Globodera spp. (e.g. G. pallida or G. rostochiensis); Rotylenchulus spp.(e.g. R. reniformis); Pratylenchus spp. (e.g. P. coffeae, P. Zeae or P.goodeyi); Radopholus spp. (e.g. R. similis); Hirschmaniella spp. (e.g.H. oryzae); Ancylostoma spp. (e.g. A. caninum, A. ceylanicum, A.duodenale or A. tubaeforme); Anisakid; Aphelenchoides spp. (e.g. A.Besseyi); Ascarids; Ascaris spp., (e.g. A. suum or A. lumbridoides);Belonolaimus spp.; Brugia spp. (e.g. B. malayi or B. pahangi);Bursaphelenchus spp.; Caenorhabditis spp. (e.g. C. elegans, C. briggsaeor C. remanei); Clostridium spp. (e.g. C. acetobutylicum); Cooperia spp.(e.g. C. oncophora); Criconemoides spp.; Cyathostomum spp. (e.g. C.catinatum, C. coronatum or C. pateratum); Cylicocyclus spp. (e.g. C.insigne, C. nassatus or C. radiatus); Cylicostephanus spp. (e.g. C.goldi or C. longibursatus); Diphyllobothrium; Dirofilaria spp. (e.g. D.immitis); Ditylenchus spp. (e.g. D. dipsaci, D. destructor or D.Angustus); Enterobius spp. (e.g. E. vermicularis); Haemonchus spp. (e.g.H. contortus); Helicotylenchus spp.; Hoplolaimus spp.; Litomosoides spp.(e.g. L. sigmodontis); Longidorus spp. (e.g. L. macrosoma); Necator spp.(e.g. N. americanus); Nippostrongylus spp. (e.g. N. brasiliensis);Onchocerca spp. (e.g. O. volvulus); Ostertagia spp. (e.g. O. ostertagi);Parastrongyloides spp. (e.g. P. trichosuri); Paratrichodorus spp. (e.g.P. minor or P. teres); Parelaphostrongylus spp. (e.g. P. tenuis);Radophulus spp.; Scutellonerna. spp.; Strongyloides spp. (e.g. S. Rattior S. stercoralis); Teladorsagia spp. (e.g. T. circumcincta); Toxascarisspp. (e.g. T. leonina); Toxocara spp. (e.g. T. canis or T. cati);Trichinella spp. (e.g. T. britovi, T. spiralis or T. spirae);Trichodorus spp. (e.g. T. similis); Trichuris spp. (e.g. T. muris, T.vulpis or T. trichiura); Iylenchulus spp.; Tylenchorhynchus spp.;Uncinaria spp. (e.g. U. stenocephala); Wuchereria spp. (e.g. W.bancrofti); Xiphinema spp. (e.g. X. Index or X. americanum).

(2) a nematode capable of infesting humans such as, but not limited to:Enterobius vermicularis, the pinworm that causes enterobiasis; Ascarislumbridoides, the large intestinal roundworm that causes ascariasis;Necator and Ancylostoma, two types of hookworms that causeancylostomiasis; Trichuris trichiura, the whipworm that causestrichuriasis; Strongyloides stercoralis that causes strongyloidiasis;and Trichonella spirae that causes trichinosis; Brugia malayi andWuchereria bancrofti, the filarial nematodes associated with the worminfections known as lymphatic filariasis and its gross manifestation,elephantiasis, and Onchocerca volvulus that causes river blindness.Transfer of nematodes to humans may also occur through blood-feedingmosquitoes which have fed upon infected animals or humans;

(3) a nematode capable of infesting animals such as, but not limited to:dogs (Hookworms e.g. Ancylostoma caninum or Uncinaria stenocephala,Ascarids e.g. Toxocara canis or Toxascaris leonina, or Whipworms e.g.Trichuris vulpis), cats (Hookworms e.g. Ancylostoma tubaeforme, Ascaridse.g. Toxocara cati), fish (herring worms or cod worms e.g. Anisakid, ortapeworm e.g. Diphyllobothrium), sheep (Wire worms e.g. Haemonchuscontortus) and cattle (Gastro-intestinal worms e.g. Ostertagiaostertagi, Cooperia oncophora);

(4) a nematode that causes unwanted damage to substrates or materials,such as nematodes that attack foodstuffs, seeds, wood, paint, plastic,clothing etc. Examples, of such nematodes include but are not limited toMeloidogyne spp. (e.g. M. incognita, M. javanica, M. arenaria, M.graminicola, M. chitwoodi or M. hapla); Heterodera spp. (e.g. H. oryzae,H. glycines, H. zeae or H. schachtii); Globodera spp. (e.g. G. pallidaor G. rostochiensis); Ditylenchus spp. (e.g. D. dipsaci, D. destructoror D. angustus); Belonolaimus spp.; Rotylenchulus spp. (e.g. R.reniformis); Pratylenchus spp. (e.g. P. coffeae, P. goodeyi or P. zeae);Radopholus spp. (e.g. R. Similis); Hirschmaniella spp. (e.g. H. oryzae);Aphelenchoides spp. (e.g. A. besseyi); Criconemoides spp.; Longidorusspp.; Helicotylenchus spp.; Hoplolaimus spp.; Xiphinema spp.;Paratrichodorus spp. (e.g. P. minor); Tylenchorhynchus spp;

(5) virus transmitting nematodes (e.g. Longidorus macrosoma: transmitsprunus necrotic ring spot virus, Xiphinema americanum: transmits tobaccoring spot virus, Paratrichadorus teres: transmits pea early browningvirus, or Trichodorus similis: transmits tobacco rattle virus).

Fungal pests of particular interest include but are not limited to thefollowing. In one embodiment of the invention, the fungus may be a mold,or more particularly a filamentous fungus. In other embodiments of theinvention, the fungus may be a yeast.

In one embodiment the fungus may be an ascomycetes fungus, i.e. a fungusbelonging to the Phylum Ascomycota.

In preferred, but non-limiting, embodiments of the invention the fungalcell is chosen from the group consisting of:

(1) a fungal cell of, or a cell derived from a plant pathogenic fungus,such as but not limited to Acremoniella spp., Alternaria spp. (e.g.Alternaria brassicola or Alternaria solani), Ascochyta spp. (e.g.Ascochyta pisi), Botrytis spp. (e.g. Botrytis cinerea or Botryotiniafuckeliana), Cladosporium spp., Cercospora spp. (e.g. Cercosporakikuchii or Cercospora zaea-maydis), Cladosporium spp. (e.g.Cladosporium fulvum), Colletotrichum spp. (e.g. Colletotrichumlindemuthianum), Curvularia spp., Diplodia spp. (e.g. Diplodia maydis),Erysiphe spp. (e.g. Erysiphe graminis f.sp. graminis, Erysiphe graminisf.sp. hordei or Erysiphe pisi), Erwinia armylovora, Fusarium spp. (e.g.Fusarium nivale, Fusarium sporotrichioides, Fusarium oxysporum, Fusariumgraminearum, Fusarium germinearum, Fusarium culmorum, Fusarium solani,Fusarium moniliforme or Fusarium roseum), Gaeumanomyces spp. (e.g.Gaeumanomyces graminis f.sp. tritici), Gibberella spp. (e.g. Gibberellazeae), Helminthosporium spp. (e.g. Helminthosporium turcicum,Helminthosporium carbonum, Helminthosporium mavdis or Helminthosporiumsigmoideum), Leptosphaeria salvinii, Macrophomina spp. (e.g.Macrophomina phaseolina), Magnaportha spp. (e.g. Magnaporthe oryzae),Mycosphaerella spp., Nectria spp. (e.g. Nectria heamatococca),Peronospora spp. (e.g. Peronospora manshurica or Peronospora tabacina),Phoma spp. (e.g. Phoma betae), Phakopsora spp. (e.g. Phakopsorapachyrhizi), Phymatotrichum spp. (e.g. Phymatotrichum omnivorum),Phytophthora spp. (e.g. Phytophthora cinnamomi, Phytophthora cactorum,Phytophthora phaseoli, Phytophthora parasitica, Phytophthoracitrophthora, Phytophthora megasperma f.sp. soiae or Phytophthorainfestans), Plasmopara spp. (e.g. Plasmopara viticola), Podosphaera spp.(e.g. Podosphaera leucotricha), Puccinia spp. (e.g. Puccinia sorghi,Puccinia striiformis, Puccinia graminis f.sp. tritici, Pucciniaasparagi, Puccinia recondita or Puccinia arachidis), Pythium spp. (e.g.Pythium aphanidermatum), Pyrenophora spp. (e.g. Pyrenophoratritici-repentens or Pyrenophora teres), Pyricularia spp. (e.g.Pyricularia oryzae), Pythium spp. (e.g. Pythium ultimum), Rhincosporiumsecalis, Rhizoctonia spp. (e.g. Rhizoctonia solani, Rhizoctonia oryzaeor Rhizoctonia cerealis), Rhizopus spp. (e.g. Rhizopus chinensid),Scerotium spp. (e.g. Scerotium rolfsii), Sclerotinia spp. (e.g.Sclerotinia sclerotiorum), Septoria spp. (e.g. Septoria lycopersici,Septoria glycines, Septoria nodorum or Septoria tritici), Thielaviopsisspp. (e.g. Thielaviopsis basicola), Tilletia spp., Trichoderma spp.(e.g. Trichoderma virde), Uncinula spp. (e.g. Uncinula necator),Ustilago maydis (e.g. corn smut), Venturia spp. (e.g. Venturiainaequalis or Venturia pirina) or Verticillium spp. (e.g. Verticilliumdahliae or Verticillium albo-atrum);

(2) a fungal cell of, or a cell derived from a fungus capable ofinfesting humans such as, but not limited to, Candida spp., particularlyCandida albicans; Dermatophytes including Epidermophyton spp.,Trichophyton spp, and Microsporum spp.; Aspergillus spp. (particularlyAspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans,Aspergillus niger or Aspergillus terreus); Blastomyces dermatitidis;Paracoccidioides brasiliensis; Coccidioides immitis; Cryptococcusneoformans; Histoplasma capsulatum Var. capsulatum or Var. duboisii;Sporothrix schenckii; Fusarium spp.; Scopulariopsis brevicaulis;Fonsecaea spp.; Penicillium spp.; or Zygomycetes group of fungi(particularly Absidia corymbifera, Rhizomucor pusillus or Rhizopusarrhizus);

(3) a fungal cell of, or a cell derived from a fungus capable ofinfesting animals such as, but not limited to Candida spp., Microsporumspp. (particularly Microsporum canis or Microsporum gypseum),Trichophyton mentagrophytes, Aspergillus spp., or Cryptococcusneoforman;

and

(4) a fungal cell of, or a cell derived from a fungus that causesunwanted damage to substrates or materials, such as fungi that attackfoodstuffs, seeds, wood, paint, plastic, clothing etc. Examples of suchfungi are the moulds, including but not limited to Stachybotrys spp.,Aspergillus spp., Alternaria spp., Cladosporium spp., Penicillium spp.or Phanerochaete chrysosporium.

II. Identification of Target Sequences

The present invention provides a method for identifying and obtaining anucleic acid comprising a nucleotide sequence for producing a dsRNA orsiRNA. For example, such a method comprises: (a) probing a cDNA orgenomic DNA library with a hybridization probe comprising all or aportion of a nucleotide sequence or a homolog thereof from a targetedpest; (b) identifying a DNA clone that hybridizes with the hybridizationprobe; (c) isolating the DNA clone identified in step (b); and (d)sequencing the cDNA or genomic DNA fragment that comprises the cloneisolated in step (c) wherein the sequenced nucleic acid moleculetranscribes all or a substantial portion of the RNA nucleotide acidsequence or a homolog thereof.

Additionally, the present invention contemplates a method for obtaininga nucleic acid fragment comprising a nucleotide sequence for producing asubstantial portion of a dsRNA or siRNA comprising: (a) synthesizingfirst and a second oligonucleotide primers corresponding to a portion ofone of the nucleotide sequences from a targeted pest; and (b) amplifyinga cDNA or genomic DNA template in a cloning vector using the first andsecond oligonucleotide primers of step (a) wherein the amplified nucleicacid molecule transcribes a substantial portion of a dsRNA or siRNA ofthe present invention.

In practicing the present invention, a target gene may be derived fromany pest that causes damage to another organism. Several criteria may beemployed in the selection of preferred target genes. The gene is onewhose protein product has a rapid turnover rate, so that dsRNAinhibition will result in a rapid decrease in protein levels. In certainembodiments it is advantageous to select a gene for which a small dropin expression level results in deleterious effects for the recipientpest. If it is desired to target a broad range of insect species, forexample, a gene is selected that is highly conserved across thesespecies. Conversely, for the purpose of conferring specificity, incertain embodiments of the invention, a gene is selected that containsregions that are poorly conserved between individual insect species, orbetween insects and other organisms. In certain embodiments it may bedesirable to select a gene that has no known homologs in otherorganisms.

As used herein, the term “derived from” refers to a specified nucleotidesequence that may be obtained from a particular specified source orspecies, albeit not necessarily directly from that specified source orspecies.

In one embodiment, a gene is selected that is expressed in the insectgut. Targeting genes expressed in the gut avoids the requirement for thedsRNA to spread within the insect. Target genes for use in the presentinvention may include, for example, those that share substantialhomologies to the nucleotide sequences of known gut-expressed genes thatencode protein components of the plasma membrane proton V-ATPase (Dow etal., 1997, Dow, 1999). This protein complex is the sole energizer ofepithelial ion transport and is responsible for alkalinization of themidgut lumen. The V-ATPase is also expressed in the Malpighian tubule,an outgrowth of the insect hindgut that functions in fluid balance anddetoxification of foreign compounds in a manner analogous to a kidneyorgan of a mammal.

In another embodiment, a gene is selected that is essentially involvedin the growth, development, and reproduction of an insect. Exemplarygenes include but are not limited to the structural subunits ofribosomal proteins and a beta-coatamer gene, CHD3 gene. Ribosomalproteins such as S4 (RpS4) and S9(RpS9) are structural constituents ofthe ribosome involved in protein biosynthesis and which are componentsof the cytosolic small ribosomal subunit, the ribosomal proteins such asL9 and L19 are structural constituent of ribosome involved in proteinbiosynthesis which is localised to the ribosome. The beta-coatamer genein C. elegans encodes a protein which is a subunit of a multimericcomplex that forms a membrane vesicle coat Similar sequences have beenfound in diverse organisms such as Arabidopsis thaliana, Drosophilamelanogaster, and Saccharomyces cerevisiae. Related sequences are foundin diverse organisms such as Leptinotarsa decemlineata, Phaedoncochleariae, Epilachna varivetis, Anthonomus grandis, Triboliumcastaneum, Myzus persicae, Nilaparvata lugens, Chilo suppressalis,Plutella xylostella and Acheta domesticus. Other target genes for use inthe present invention may include, for example, those that playimportant roles in viability, growth, development, reproduction, andinfectivity. These target genes include, for example, house keepinggenes, transcription factors, and insect specific genes or lethalknockout mutations in Caenorhabditis or Drosophila. The target genes foruse in the present invention may also be those that are from otherorganisms, e.g., from a nematode (e.g., Meloidogyne spp. or Heteroderaspp.), other insects or arachnidae (e.g. Leptinotarsa spp., Phaedonspp., Epilachna spp., Anthonomus spp., Tribolium spp., Myzus spp.,Nilaparvata spp., Chilo spp., Plutella spp., or Acheta spp.Additionally, the nucleotide sequences for use as a target sequence inthe present invention may also be derived from viral, bacterial, fungal,insect or fungal genes whose functions have been established fromliterature and the nucleotide sequences of which share substantialsimilarity with the target genes in the genome of an insect.

For many of the insects that are potential targets for control by thepresent invention, there may be limited information regarding thesequences of most genes or the phenotype resulting from mutation ofparticular genes. Therefore, genes may be selected based on availableinformation available concerning corresponding genes in a modelorganism, such as Caenorhabditis or Drosophila, or in some other insectspecies. Genes may also be selected based on available sequenceinformation for other species, such as nematode or fungal species, inwhich the genes have been characterized. In some cases it will bepossible to obtain the sequence of a corresponding gene from a targetinsect by searching databases, such as GenBank, using either the name ofthe gene or the gene sequence. Once the sequence is obtained, PCR may beused to amplify an appropriately selected segment of the gene in theinsect for use in the present invention.

In order to obtain a DNA segment from the corresponding gene in aninsect species, for example, PCR primers may be designed based on thesequence as found in C. elegans or Drosophila, or an insect from whichthe gene has already been cloned. The primers are designed to amplify aDNA segment of sufficient length for use in the present invention.Amplification conditions are selected so that amplification will occureven if the primers do not exactly match the target sequence.Alternately, the gene, or a portion thereof, may be cloned from agenomic DNA or cDNA library prepared from the insect pest species, usinga known insect gene as a probe. Techniques for performing PCR andcloning from libraries are known. Further details of the process bywhich DNA segments from target insect pest species may be isolated basedon the sequence of genes previously cloned from an insect species areprovided in the Examples. One of ordinary skill in the art willrecognize that a variety of techniques may be used to isolate genesegments from insect pest species that correspond to genes previouslyisolated from other species.

III. Methods for Inhibiting or Suppressing a Target Gene

The present invention provides methods for inhibiting gene expression ofone or multiple target genes in a target pest using stabilized dsRNAmethods. The invention is particularly useful for modulating eukaryoticgene expression, in particular modulating the expression of genespresent in pests that exhibit a digestive system pH level that is fromabout 4.5 to about 9.5, more preferably from about 5.0 to about 8.0, andeven more preferably from about 6.5 to about 7.5. For pests with adigestive system that exhibits pH levels outside of these ranges,delivery methods may be desired for use that do not require ingestion ofdsRNA molecules.

The methods of the invention encompass the simultaneous or sequentialprovision of two or more different double-stranded RNAs or RNAconstructs to the same insect, so as to achieve down-regulation orinhibition of multiple target genes or to achieve a more potentinhibition of a single target gene.

Alternatively, multiple targets are hit by the provision of onedouble-stranded RNA that hits multiple target sequences, and a singletarget is more efficiently inhibited by the presence of more than onecopy of the double stranded RNA fragment corresponding to the targetgene. Thus, in one embodiment of the invention, the double-stranded RNAconstruct comprises multiple dsRNA regions, at least one strand of eachdsRNA region comprising a nucleotide sequence that is complementary toat least part of a target nucleotide sequence of an insect target gene.According to the invention, the dsRNA regions in the RNA construct maybe complementary to the same or to different target genes and/or thedsRNA regions may be complementary to targets from the same or fromdifferent insect species. Use of such dsRNA constructs in a plant hostcell, thus establishes a more potent resistance to a single or tomultiple insect species in the plant. In one embodiment, the doublestranded RNA region comprises multiple copies of the nucleotide sequencethat is complementary to the target gene. Alternatively, the dsRNA hitsmore than one target sequence of the same target gene. The inventionthus encompasses isolated double stranded RNA constructs comprising atleast two copies of said nucleotide sequence complementary to at leastpart of a nucleotide sequence of an insect target. DsRNA that hits morethan one of the above-mentioned targets, or a combination of differentdsRNA against different of the above mentioned targets are developed andused in the methods of the present invention. Suitable dsRNA nucleotidesand dsRNA constructs are described in WO2006/046148 by applicant, whichis incorporated herein in its entirety.

The terms “hit”, “hits”, and “hitting” are alternative wordings toindicate that at least one of the strands of the dsRNA is complementaryto, and as such may bind to, the target gene or nucleotide sequence.

The modulatory effect of dsRNA is applicable to a variety of genesexpressed in the pests including, for example, endogenous genesresponsible for cellular metabolism or cellular transformation,including house keeping genes, transcription factors, and other geneswhich encode polypeptides involved in cellular metabolism.

As used herein, the phrase “inhibition of gene expression” or“inhibiting expression of a target gene in the cell of an pest” refersto the absence (or observable decrease) in the level of protein and/ormRNA product from the target gene. Specificity refers to the ability toinhibit the target gene without manifest effects on other genes of thecell and without any effects on any gene within the cell that isproducing the dsRNA molecule. The inhibition of gene expression of thetarget gene in the pest may result in novel phenotypic traits in thepest.

“Gene suppression” refers to any of the well-known methods for reducingthe levels of gene transcription to mRNA and/or subsequent translationof the mRNA. Gene suppression is also intended to mean the reduction ofprotein expression from a gene or a coding sequence includingposttranscriptional gene suppression and transcriptional suppression.Posttranscriptional gene suppression is mediated by the homology betweenof all or a part of a mRNA transcribed from a gene or coding sequencetargeted for suppression and the corresponding double stranded RNA usedfor suppression, and refers to the substantial and measurable reductionof the amount of available mRNA available in the cell for binding byribosomes. The transcribed RNA can be in the sense orientation to effectwhat is called co-suppression, in the anti-sense orientation to effectwhat is called anti-sense suppression, or in both orientations producinga dsRNA to effect what is called RNA interference (RNAi).

Transcriptional suppression is mediated by the presence in the cell of adsRNA gene suppression agent exhibiting substantial sequence identity toa promoter DNA sequence or the complement thereof to effect what isreferred to as promoter trans suppression. Gene suppression may beeffective against a native host gene associated with a trait, e.g., toprovide hosts with reduced levels of a protein encoded by the nativegene or with enhanced or reduced levels of an affected metabolite. Genesuppression can also be effective against target genes in pests that mayingest or contact material containing gene suppression agents,specifically designed to inhibit or suppress the expression of one ormore homologous or complementary sequences in the cells of the pest.

A beneficial method of post transcriptional gene suppression in hostsemploys both sense-oriented and anti-sense-oriented, transcribed RNAwhich is stabilized, e.g., as a hairpin and stem and loop structure. Apreferred DNA construct for effecting post transcriptional genesuppression is one in which a first segment encodes an RNA exhibiting ananti-sense orientation exhibiting substantial identity to a segment of agene targeted for suppression, which is linked to a second segment insense orientation encoding an RNA exhibiting substantial complementarityto the first segment. Such a construct forms a stem and loop structureby hybridization of the first segment with the second segment and a loopstructure from the nucleotide sequences linking the two segments (seeWO94/01550, WO98/05770, US 2002/0048814, and US 2003/0018993).

According to one embodiment of the present invention, there is provideda nucleotide sequence, for which in vitro expression results intranscription of a stabilized RNA sequence that is substantiallyhomologous to an RNA molecule of a targeted gene in a pest thatcomprises an RNA sequence encoded by a nucleotide sequence within thegenome of the pest. Thus, after the pest uptakes the stabilized RNAsequence, or is otherwise exposed to the dsRNA, a down-regulation of thenucleotide sequence corresponding to the target gene in the cells of atarget pest is affected.

Inhibition of a target gene using the stabilized dsRNA technology of thepresent invention is sequence-specific in that nucleotide sequencescorresponding to the duplex region of the RNA are targeted for geneticinhibition. RNA containing a nucleotide sequences identical to a portionof the target gene is preferred for inhibition. RNA sequences withinsertions, deletions, and single point mutations relative to the targetsequence have also been found to be effective for inhibition. Inperformance of the present invention, it is preferred that theinhibitory dsRNA and the portion of the target gene share at least fromabout 80% sequence identity, or from about 85% sequence identity, orfrom about 90% sequence identity, or from about 95% sequence identity,or from about 99% sequence identity, or even about 100% sequenceidentity. Alternatively, the duplex region of the RNA may be definedfunctionally as a nucleotide sequence that is capable of hybridizingwith a portion of the target gene transcript. A less than full lengthsequence exhibiting a greater homology compensates for a longer lesshomologous sequence. The length of the identical nucleotide sequencesmay be at least about 25, 50, 100, 200, 300, 400, 500 or at least about1000 bases. Normally, a sequence of greater than 20-100 nucleotidesshould be used, though a sequence of greater than about 200-300nucleotides would be preferred, and a sequence of greater than about500-1000 nucleotides would be especially preferred depending on the sizeof the target gene. The invention has the advantage of being able totolerate sequence variations that might be expected due to geneticmutation, strain polymorphism, or evolutionary divergence. Theintroduced nucleic acid molecule may not need to be absolute homology,may not need to be full length, relative to either the primarytranscription product or fully processed mRNA of the target gene.Therefore, those skilled in the art need to realize that, as disclosedherein, 100% sequence identity between the RNA and the target gene isnot required to practice the present invention.

IV. Methods for Preparing dsRNA

dsRNA molecules may be synthesized either in vivo or in vitro. The dsRNAmay be formed by a single self-complementary RNA strand or from twocomplementary RNA strands. Endogenous RNA polymerase of the cell maymediate transcription in vivo, or cloned RNA polymerase can be used fortranscription in vivo or in vitro. Inhibition may be targeted byspecific transcription in an organ, tissue, or cell type; stimulation ofan environmental condition (e.g., infection, stress, temperature,chemical inducers); and/or engineering transcription at a developmentalstage or age. The RNA strands may or may not be polyadenylated; the RNAstrands may or may not be capable of being translated into a polypeptideby a cell's translational apparatus.

A RNA, dsRNA, siRNA, or miRNA of the present invention may be producedchemically or enzymatically by one skilled in the art through manual orautomated reactions or in vivo in another organism. RNA may also beproduced by partial or total organic synthesis; any modifiedribonucleotide can be introduced by in vitro enzymatic or organicsynthesis. The RNA may be synthesized by a cellular RNA polymerase or abacteriophage RNA polymerase (e.g., T3, T7, SP6). The use and productionof an expression construct are known in the art (see, for example, WO97/32016; U.S. Pat. Nos. 5,593,874, 5,698,425, 5,712,135, 5,789,214, and5,804,693). If synthesized chemically or by in vitro enzymaticsynthesis, the RNA may be purified prior to introduction into the cell.For example, RNA can be purified from a mixture by extraction with asolvent or resin, precipitation, electrophoresis, chromatography, or acombination thereof. Alternatively, the RNA may be used with no or aminimum of purification to avoid losses due to sample processing. TheRNA may be dried for storage or dissolved in an aqueous solution. Thesolution may contain buffers or salts to promote annealing, and/orstabilization of the duplex strands.

V. Polynucleotide Sequences

Provided according to the invention are nucleotide sequences, theexpression of which results in an RNA sequence which is substantiallyhomologous to an RNA molecule of a targeted gene in a pest thatcomprises an RNA sequence encoded by a nucleotide sequence within thegenome of the pest. Thus, after ingestion of the dsRNA sequencedown-regulation of the nucleotide sequence of the target gene in thecells of the pest may be obtained resulting in a deleterious effect onthe maintenance, viability, proliferation, reproduction, and infestationof the pest.

Each “nucleotide, sequence” set forth herein is presented as a sequenceof deoxyribonucleotides (abbreviated A, G, C and T). However, by“nucleotide sequence” of a nucleic acid molecule or polynucleotide isintended, for a DNA molecule or polynucleotide, a sequence ofdeoxyribonucleotides, and for an RNA molecule or polynucleotide, thecorresponding sequence of ribonucleotides (A, G, C and U) where eachthymidine deoxynucleotide (T) in the specified deoxynucleotide sequencein is replaced by the ribonucleotide uridine (U).

As used herein, “nucleic acid” refers to a single or double-strandedpolymer of deoxyribonucleotide or ribonucleotide bases read from the 5′to the 3′ end. A nucleic acid may also optionally contain non-naturallyoccurring or altered nucleotide bases that permit correct read throughby a polymerase and do not reduce expression of a polypeptide encoded bythat nucleic acid. “Nucleotide sequence” or “nucleic acid sequence”refers to both the sense and antisense strands of a nucleic acid aseither individual single strands or in the duplex.

The term “ribonucleic acid” (RNA) is inclusive of RNAi (inhibitory RNA),dsRNA (double stranded RNA), siRNA (small interfering RNA), mRNA(messenger RNA), miRNA (micro-RNA), tRNA (transfer RNA, whether chargedor discharged with a corresponding acylated amino acid), and cRNA(complementary RNA) and the term “deoxyribonucleic acid” (DNA) isinclusive of cDNA and genomic DNA and DNA-RNA hybrids.

The words “nucleic acid segment”, “nucleotide sequence segment”, or moregenerally “segment” will be understood by those in the art as afunctional term that includes both genomic sequences, ribosomal RNAsequences, transfer RNA sequences, messenger RNA sequences, operonsequences and smaller engineered nucleotide sequences that express ormay be adapted to express, proteins, polypeptides or peptides.

Accordingly, the present invention relates to an isolated nucleicmolecule comprising a polynucleotide having a sequence selected from thegroup consisting of any of the polynucleotide sequences of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 49-158, 159, 160, 163, 168,173, 178, 183, 188, 193, 198, 203, 208, 215, 220, 225, 230, 247, 249,251, 253, 255, 257, 259, 275-472, 473, 478, 483, 488, 493, 498, 503,513, 515, 517, 519, 521, 533-575, 576, 581, 586, 591, 596, 601, 603,605, 607, 609, 621-767, 768, 773, 778, 783, 788, 793, 795, 797, 799,801, 813-862, 863, 868, 873, 878, 883, 888, 890, 892, 894, 896,908-1040, 1041, 1046, 1051, 1056, 1061, 1071, 1073, 1075, 1077, 1079,1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099, 1101, 1103,1105, 1107, 1109, 1111, 1113, 1161-1571, 1572, 1577, 1582, 1587, 1592,1597, 1602, 1607, 1612, 1617, 1622, 1627, 1632, 1637, 1642, 1647, 1652,1657, 1662, 1667, 1672, 1677, 1682, 1684, 1686, 1688, 1690, 1692, 1694,1696, 1698, 1700, 1702, 1704, 1730-2039, 2040, 2045, 2050, 2055, 2060,2065, 2070, 2075, 2080, 2085, 2090, 2095, 2100, 2102, 2104, 2106, 2108,2120-2338, 2339, 2344, 2349, 2354, 2359, 2364, 2366, 2368, 2370, 2372,2384-2460, 2461, 2466, 2471, 2476 and 2481. The invention also providesfunctional fragments of the polynucleotide sequences of SEQ ID NOs: 1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 49-158, 159, 160, 163, 168, 173,178, 183, 188, 193, 198, 203, 208, 215, 220, 225, 230, 247, 249, 251,253, 255, 257, 259, 275-472, 473, 478, 483, 488, 493, 498, 503, 513,515, 517, 519, 521, 533-575, 576, 581, 586, 591, 596, 601, 603, 605,607, 609, 621-767, 768, 773, 778, 783, 788, 793, 795, 797, 799, 801,813-862, 863, 868, 873, 878, 883, 888, 890, 892, 894, 896, 908-1040,1041, 1046, 1051, 1056, 1061, 1071, 1073, 1075, 1077, 1079, 1081, 1083,1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099, 1101, 1103, 1105, 1107,1109, 1111, 1113, 1161-1571, 1572, 1577, 1582, 1587, 1592, 1597, 1602,1607, 1612, 1617, 1622, 1627, 1632, 1637, 1642, 1647, 1652, 1657, 1662,1667, 1672, 1677, 1682, 1684, 1686, 1688, 1690, 1692, 1694, 1696, 1698,1700, 1702, 1704, 1730-2039, 2040, 2045, 2050, 2055, 2060, 2065, 2070,2075, 2080, 2085, 2090, 2095, 2100, 2102, 2104, 2106, 2108, 2120-2338,2339, 2344, 2349, 2354, 2359, 2364, 2366, 2368, 2370, 2372, 2384-2460,2461, 2466, 2471, 2476 and 2481. The invention further providescomplementary nucleic acids, or fragments thereof, to any of thepolynucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 49-158, 159, 160, 163, 168, 173, 178, 183, 188, 193, 198,203, 208, 215, 220, 225, 230, 247, 249, 251, 253, 255, 257, 259,275-472, 473, 478, 483, 488, 493, 498, 503, 513, 515, 517, 519, 521,533-575, 576, 581, 586, 591, 596, 601, 603, 605, 607, 609, 621-767, 768,773, 778, 783, 788, 793, 795, 797, 799, 801, 813-862, 863, 868, 873,878, 883, 888, 890, 892, 894, 896, 908-1040, 1041, 1046, 1051, 1056,1061, 1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091,1093, 1095, 1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113,1161-1571, 1572, 1577, 1582, 1587, 1592, 1597, 1602, 1607, 1612, 1617,1622, 1627, 1632, 1637, 1642, 1647, 1652, 1657, 1662, 1667, 1672, 1677,1682, 1684, 1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704,1730-2039, 2040, 2045, 2050, 2055, 2060, 2065, 2070, 2075, 2080, 2085,2090, 2095, 2100, 2102, 2104, 2106, 2108, 2120-2338, 2339, 2344, 2349,2354, 2359, 2364, 2366, 2368, 2370, 2372, 2384-2460, 2461, 2466, 2471,2476 and 2481, as well as a nucleic acid, comprising at least 15contiguous bases, which hybridizes to any of the polynucleotidesequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,49-158, 159, 160, 163, 168, 173, 178, 183, 188, 193, 198, 203, 208, 215,220, 225, 230, 247, 249, 251, 253, 255, 257, 259, 275-472, 473, 478,483, 488, 493, 498, 503, 513, 515, 517, 519, 521, 533-575, 576, 581,586, 591, 596, 601, 603, 605, 607, 609, 621-767, 768, 773, 778, 783,788, 793, 795, 797, 799, 801, 813-862, 863, 868, 873, 878, 883, 888,890, 892, 894, 896, 908-1040, 1041, 1046, 1051, 1056, 1061, 1071, 1073,1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097,1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113, 1161-1571, 1572, 1577,1582, 1587, 1592, 1597, 1602, 1607, 1612, 1617, 1622, 1627, 1632, 1637,1642, 1647, 1652, 1657, 1662, 1667, 1672, 1677, 1682, 1684, 1686, 1688,1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704, 1730-2039, 2040, 2045,2050, 2055, 2060, 2065, 2070, 2075, 2080, 2085, 2090, 2095, 2100, 2102,2104, 2106, 2108, 2120-2338, 2339, 2344, 2349, 2354, 2359, 2364, 2366,2368, 2370, 2372, 2384-2460, 2461, 2466, 2471, 2476 and 2481.

The present invention also provides orthologous sequences, andcomplements and fragments thereof, of the polynucleotide sequences ofSEQ D NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 49-158, 159, 160,163, 168, 173, 178, 183, 188, 193, 198, 203, 208, 215, 220, 225, 230,247, 249, 251, 253, 255, 257, 259, 275-472, 473, 478, 483, 488, 493,498, 503, 513, 515, 517, 519, 521, 533-575, 576, 581, 586, 591, 596,601, 603, 605, 607, 609, 621-767, 768, 773, 778, 783, 788, 793, 795,797, 799, 801, 813-862, 863, 868, 873, 878, 883, 888, 890, 892, 894,896, 908-1040, 1041, 1046, 1051, 1056, 1061, 1071, 1073, 1075, 1077,1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099, 1101,1103, 1105, 1107, 1109, 1111, 1113, 1161-1571, 1572, 1577, 1582, 1587,1592, 1597, 1602, 1607, 1612, 1617, 1622, 1627, 1632, 1637, 1642, 1647,1652, 1657, 1662, 1667, 1672, 1677, 1682, 1684, 1686, 1688, 1690, 1692,1694, 1696, 1698, 1700, 1702, 1704, 1730-2039, 2040, 2045, 2050, 2055,2060, 2065, 2070, 2075, 2080, 2085, 2090, 2095, 2100, 2102, 2104, 2106,2108, 2120-2338, 2339, 2344, 2349, 2354, 2359, 2364, 2366, 2368, 2370,2372, 2384-2460, 2461, 2466, 2471, 2476 and 2481 of the invention.Accordingly, the invention encompasses target genes which are insectorthologs of a gene comprising a nucleotide sequence as represented inany of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 49-158,159, 160, 163, 168, 173, 178, 183, 188, 193, 198, 203, 208, 215, 220,225, 230, 247, 249, 251, 253, 255, 257, 259, 275-472, 473, 478, 483,488, 493, 498, 503, 513, 515, 517, 519, 521, 533-575, 576, 581, 586,591, 596, 601, 603, 605, 607, 609, 621-767, 768, 773, 778, 783, 788,793, 795, 797, 799, 801, 813-862, 863, 868, 873, 878, 883, 888, 890,892, 894, 896, 908-1040, 1041, 1046, 1051, 1056, 1061, 1071, 1073, 1075,1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099,1101, 1103, 1105, 1107, 1109, 1111, 1113, 1161-1571, 1572, 1577, 1582,1587, 1592, 1597, 1602, 1607, 1612, 1617, 1622, 1627, 1632, 1637, 1642,1647, 1652, 1657, 1662, 1667, 1672, 1677, 1682, 1684, 1686, 1688, 1690,1692, 1694, 1696, 1698, 1700, 1702, 1704, 1730-2039, 2040, 2045, 2050,2055, 2060, 2065, 2070, 2075, 2080, 2085, 2090, 2095, 2100, 2102, 2104,2106, 2108, 2120-2338, 2339, 2344, 2349, 2354, 2359, 2364, 2366, 2368,2370, 2372, 2384-2460, 2461, 2466, 2471, 2476 and 2481. By way ofexample, insect orthologues may comprise a nucleotide sequence asrepresented in any of SEQ ID NOs: 49-123, 275-434, 533-562, 621-738,813-852, 908-1010, 1161-1437, 1730-1987, 2120-2290, 2384-2438, or afragment thereof of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26 or 27 nucleotides. A non-limiting list of insect or arachnidaorthologs genes or sequences comprising at least a fragment of 15,preferably at least 17 bp of one of the sequences of the invention isgiven in Tables 4.

The invention also encompasses target genes which are nematode orthologsof a gene comprising a nucleotide sequence as represented in any of SEQID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 49-158, 159, 160,163, 168, 173, 178, 183, 188, 193, 198, 203, 208, 215, 220, 225, 230,247, 249, 251, 253, 255, 257, 259, 275-472, 473, 478, 483, 488, 493,498, 503, 513, 515, 517, 519, 521, 533-575, 576, 581, 586, 591, 596,601, 603, 605, 607, 609, 621-767, 768, 773, 778, 783, 788, 793, 795,797, 799, 801, 813-862, 863, 868, 873, 878, 883, 888, 890, 892, 894,896, 908-1040, 1041, 1046, 1051, 1056, 1061, 1071, 1073, 1075, 1077,1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099, 1101,1103, 1105, 1107, 1109, 1111, 1113, 1161-1571, 1572, 1577, 1582, 1587,1592, 1597, 1602, 1607, 1612, 1617, 1622, 1627, 1632, 1637, 1642, 1647,1652, 1657, 1662, 1667, 1672, 1677, 1682, 1684, 1686, 1688, 1690, 1692,1694, 1696, 1698, 1700, 1702, 1704, 1730-2039, 2040, 2045, 2050, 2055,2060, 2065, 2070, 2075, 2080, 2085, 2090, 2095, 2100, 2102, 2104, 2106,2108, 2120-2338, 2339, 2344, 2349, 2354, 2359, 2364, 2366, 2368, 2370,2372, 2384-2460, 2461, 2466, 2471, 2476, and 2481 of the invention. Byway of example, nematode orthologs may comprise a nucleotide sequence asrepresented in any of SEQ ID NOs: 124-135, 435-446, 563, 564, 739-751,853, 854, 1011-1025, 1438-1473, 1988-2001, 2291-2298, 2439-2440 of theinvention, or a fragment of at least 15, 16, 17, 18, 19, 20 or 21nucleotides thereof. According to another aspect, the invention thusencompasses any of the methods described herein for controlling nematodegrowth in an organism, or for preventing nematode infestation of anorganism susceptible to nematode infection, comprising contactingnematode cells with a double-stranded RNA, wherein the double-strandedRNA comprises annealed complementary strands, one of which has anucleotide sequence which is complementary to at least part of thenucleotide sequence of a target gene comprising a fragment of at least17, 18, 19, 20 or 21 nucleotides of any of the sequences as representedin SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 49-158, 159,160, 163, 168, 173, 178, 183, 188, 193, 198, 203, 208, 215, 220, 225,230, 247, 249, 251, 253, 255, 257, 259, 275-472, 473, 478, 483, 488,493, 498, 503, 513, 515, 517, 519, 521, 533-575, 576, 581, 586, 591,596, 601, 603, 605, 607, 609, 621-767, 768, 773, 778, 783, 788, 793,795, 797, 799, 801, 813-862, 863, 868, 873, 878, 883, 888, 890, 892,894, 896, 908-1040, 1041, 1046, 1051, 1056, 1061, 1071, 1073, 1075,1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099,1101, 1103, 1105, 1107, 1109, 1111, 1113, 1161-1571, 1572, 1577, 1582,1587, 1592, 1597, 1602, 1607, 1612, 1617, 1622, 1627, 1632, 1637, 1642,1647, 1652, 1657, 1662, 1667, 1672, 1677, 1682, 1684, 1686, 1688, 1690,1692, 1694, 1696, 1698, 1700, 1702, 1704, 1730-2039, 2040, 2045, 2050,2055, 2060, 2065, 2070, 2075, 2080, 2085, 2090, 2095, 2100, 2102, 2104,2106, 2108, 2120-2338, 2339, 2344, 2349, 2354, 2359, 2364, 2366, 2368,2370, 2372, 2384-2460, 2461, 2466, 2471, 2476 and 2481, whereby thedouble-stranded RNA is taken up by the fungus and thereby controlsgrowth or prevents infestation. The invention also relates tonematode-resistant transgenic plants comprising a fragment of at least17, 18, 19, 20 or 21 nucleotides of any of the sequences as representedin SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 49-158, 159,160, 163, 168, 173, 178, 183, 188, 193, 198, 203, 208, 215, 220, 225,230, 247, 249, 251, 253, 255, 257, 259, 275-472, 473, 478, 483, 488,493, 498, 503, 513, 515, 517, 519, 521, 533-575, 576, 581, 586, 591,596, 601, 603, 605, 607, 609, 621-767, 768, 773, 778, 783, 788, 793,795, 797, 799, 801, 813-862, 863, 868, 873, 878, 883, 888, 890, 892,894, 896, 908-1040, 1041, 1046, 1051, 1056, 1061, 1071, 1073, 1075,1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099,1101, 1103, 1105, 1107, 1109, 1111, 1113, 1161-1571, 1572, 1577, 1582,1587, 1592, 1597, 1602, 1607, 1612, 1617, 1622, 1627, 1632, 1637, 1642,1647, 1652, 1657, 1662, 1667, 1672, 1677, 1682, 1684, 1686, 1688, 1690,1692, 1694, 1696, 1698, 1700, 1702, 1704, 1730-2039, 2040, 2045, 2050,2055, 2060, 2065, 2070, 2075, 2080, 2085, 2090, 2095, 2100, 2102, 2104,2106, 2108, 2120-2338, 2339, 2344, 2349, 2354, 2359, 2364, 2366, 2368,2370, 2372, 2384-2460, 2461, 2466, 2471, 2476 and 2481. A non-limitinglist of nematode orthologs genes or sequences comprising at least afragment of 15, preferably at least 17 bp of one of the sequences of theinvention is given in Tables 5.

According to another embodiment, the invention encompasses target geneswhich are fungal orthologs of a gene comprising a nucleotide sequence asrepresented in any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 49-158, 159, 160, 163, 168, 173, 178, 183, 188, 193, 198, 203, 208,215, 220, 225, 230, 247, 249, 251, 253, 255, 257, 259, 275-472, 473,478, 483, 488, 493, 498, 503, 513, 515, 517, 519, 521, 533-575, 576,581, 586, 591, 596, 601, 603, 605, 607, 609, 621-767, 768, 773, 778,783, 788, 793, 795, 797, 799, 801, 813-862, 863, 868, 873, 878, 883,888, 890, 892, 894, 896, 908-1040, 1041, 1046, 1051, 1056, 1061, 1071,1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095,1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113, 1161-1571, 1572,1577, 1582, 1587, 1592, 1597, 1602, 1607, 1612, 1617, 1622, 1627, 1632,1637, 1642, 1647, 1652, 1657, 1662, 1667, 1672, 1677, 1682, 1684, 1686,1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704, 1730-2039, 2040,2045, 2050, 2055, 2060, 2065, 2070, 2075, 2080, 2085, 2090, 2095, 2100,2102, 2104, 2106, 2108, 2120-2338, 2339, 2344, 2349, 2354, 2359, 2364,2366, 2368, 2370, 2372, 2384-2460, 2461, 2466, 2471, 2476 and 2481 ofthe invention. By way of example, fungal orthologs may comprise anucleotide sequence as represented in any of SEQ ID NOs:136-158,447-472, 565-575, 752-767, 855-862, 1026-1040, 1474-1571, 2002-2039,2299-2338, 2441-2460, or a fragment of at least 17, 18, 19, 20, 21, 22,23, 24, 25, 26 or 27 nucleotides thereof. According to another aspect,the invention thus encompasses any of the methods described herein forcontrolling fungal growth on a cell or an organism, or for preventingfungal infestation of a cell or an organism susceptible to fungalinfection, comprising contacting fungal cells with a double-strandedRNA, wherein the double-stranded RNA comprises annealed complementarystrands, one of which has a nucleotide sequence which is complementaryto at least part of the nucleotide sequence of a target gene comprisinga fragment of at least 17, 18, 19, 20 or 21 nucleotides of any of thesequences as represented in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 49-158, 159, 160, 163, 168, 173, 178, 183, 188, 193, 198,203, 208, 215, 220, 225, 230, 247, 249, 251, 253, 255, 257, 259,275-472, 473, 478, 483, 488, 493, 498, 503, 513, 515, 517, 519, 521,533-575, 576, 581, 586, 591, 596, 601, 603, 605, 607, 609, 621-767, 768,773, 778, 783, 788, 793, 795, 797, 799, 801, 813-862, 863, 868, 873,878, 883, 888, 890, 892, 894, 896, 908-1040, 1041, 1046, 1051, 1056,1061, 1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091,1093, 1095, 1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113,1161-1571, 1572, 1577, 1582, 1587, 1592, 1597, 1602, 1607, 1612, 1617,1622, 1627, 1632, 1637, 1642, 1647, 1652, 1657, 1662, 1667, 1672, 1677,1682, 1684, 1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704,1730-2039, 2040, 2045, 2050, 2055, 2060, 2065, 2070, 2075, 2080, 2085,2090, 2095, 2100, 2102, 2104, 2106, 2108, 2120-2338, 2339, 2344, 2349,2354, 2359, 2364, 2366, 2368, 2370, 2372, 2384-2460, 2461, 2466, 2471,2476 and 2481, whereby the double-stranded RNA is taken up by the fungusand thereby controls growth or prevents infestation. The invention alsorelates to fungal-resistant transgenic plants comprising a fragment ofat least 17, 18, 19, 20 or 21 of any of the sequences as represented inSEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 49-158, 159, 160,163, 168, 173, 178, 183, 188, 193, 198, 203, 208, 215, 220, 225, 230,247, 249, 251, 253, 255, 257, 259, 275-472, 473, 478, 483, 488, 493,498, 503, 513, 515, 517, 519, 521, 533-575, 576, 581, 586, 591, 596,601, 603, 605, 607, 609, 621-767, 768, 773, 778, 783, 788, 793, 795,797, 799, 801, 813-862, 863, 868, 873, 878, 883, 888, 890, 892, 894,896, 908-1040, 1041, 1046, 1051, 1056, 1061, 1071, 1073, 1075, 1077,1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099, 1101,1103, 1105, 1107, 1109, 1111, 1113, 1161-1571, 1572, 1577, 1582, 1587,1592, 1597, 1602, 1607, 1612, 1617, 1622, 1627, 1632, 1637, 1642, 1647,1652, 1657, 1662, 1667, 1672, 1677, 1682, 1684, 1686, 1688, 1690, 1692,1694, 1696, 1698, 1700, 1702, 1704, 1730-2039, 2040, 2045, 2050, 2055,2060, 2065, 2070, 2075, 2080, 2085, 2090, 2095, 2100, 2102, 2104, 2106,2108, 2120-2338, 2339, 2344, 2349, 2354, 2359, 2364, 2366, 2368, 2370,2372, 2384-2460, 2461, 2466, 2471, 2476 and 2481. A non-limiting list offungal orthologs genes or sequences comprising at least a fragment of15, preferably at least 17 bp of one of the sequences of the inventionis given in Tables 6.

In a further embodiment, a dsRNA molecule of the invention comprises anyof SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 49-158, 159,160, 163, 168, 173, 178, 183, 188, 193, 198, 203, 208, 215, 220, 225,230, 247, 249, 251, 253, 255, 257, 259, 275-472, 473, 478, 483, 488,493, 498, 503, 513, 515, 517, 519, 521, 533-575, 576, 581, 586, 591,596, 601, 603, 605, 607, 609, 621-767, 768, 773, 778, 783, 788, 793,795, 797, 799, 801, 813-862, 863, 868, 873, 878, 883, 888, 890, 892,894, 896, 908-1040, 1041, 1046, 1051, 1056, 1061, 1071, 1073, 1075,1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099,1101, 1103, 1105, 1107, 1109, 1111, 1113, 1161-1571, 1572, 1577, 1582,1587, 1592, 1597, 1602, 1607, 1612, 1617, 1622, 1627, 1632, 1637, 1642,1647, 1652, 1657, 1662, 1667, 1672, 1677, 1682, 1684, 1686, 1688, 1690,1692, 1694, 1696, 1698, 1700, 1702, 1704, 1730-2039, 2040, 2045, 2050,2055, 2060, 2065, 2070, 2075, 2080, 2085, 2090, 2095, 2100, 2102, 2104,2106, 2108, 2120-2338, 2339, 2344, 2349, 2354, 2359, 2364, 2366, 2368,2370, 2372, 2384-2460, 2461, 2466, 2471, 2476 and 2481, though thesequences set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 49-158, 159, 160, 163, 168, 173, 178, 183, 188, 193, 198, 203,208, 215, 220, 225, 230, 247, 249, 251, 253, 255, 257, 259, 275-472,473, 478, 483, 488, 493, 498, 503, 513, 515, 517, 519, 521, 533-575,576, 581, 586, 591, 596, 601, 603, 605, 607, 609, 621-767, 768, 773,778, 783, 788, 793, 795, 797, 799, 801, 813-862, 863, 868, 873, 878,883, 888, 890, 892, 894, 896, 908-1040, 1041, 1046, 1051, 1056, 1061,1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093,1095, 1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113, 1161-1571,1572, 1577, 1582, 1587, 1592, 1597, 1602, 1607, 1612, 1617, 1622, 1627,1632, 1637, 1642, 1647, 1652, 1657, 1662, 1667, 1672, 1677, 1682, 1684,1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704, 1730-2039,2040, 2045, 2050, 2055, 2060, 2065, 2070, 2075, 2080, 2085, 2090, 2095,2100, 2102, 2104, 2106, 2108, 2120-2338, 2339, 2344, 2349, 2354, 2359,2364, 2366, 2368, 2370, 2372, 2384-2460, 2461, 2466, 2471, 2476 and 2481are not limiting. A dsRNA molecule of the invention can comprise anycontiguous target gene from a pest species (e.g., about 15 to about 25or more, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or morecontiguous nucleotides).

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, recombinant DNA molecules contained in a DNAconstruct are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vitro RNA transcripts of the DNAmolecules of the present invention. Isolated nucleic acid molecules,according to the present invention, further include such moleculesproduced synthetically.

Nucleic acid molecules of the present invention may be in the form ofRNA, such as mRNA, or in the form of DNA, including, for instance, cDNAand genomic DNA obtained by cloning or produced synthetically. The DNAor RNA may be double-stranded or single-stranded. Single-stranded DNAmay be the coding strand, also known as the sense strand, or it may bethe non-coding strand, also referred to as the anti-sense strand.

VI. Sequence Analysis

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373 from Applied Biosystems, Inc.).Therefore, as is known in the art for any DNA sequence determined bythis automated approach, any nucleotide sequence determined herein maycontain some errors. Nucleotide sequences determined by automation aretypically at least about 95% identical, more typically at least about96% to at least about 99.9% identical to the actual nucleotide sequenceof the sequenced DNA molecule. The actual sequence can be more preciselydetermined by other approaches including manual DNA sequencing methodswell known in the art. As is also known in the art, a single insertionor deletion in a determined nucleotide sequence compared to the actualsequence will cause a frame shift in translation of the nucleotidesequence such that the predicted amino acid sequence encoded by adetermined nucleotide sequence may be completely different from theamino acid sequence actually encoded by the sequenced DNA molecule,beginning at the point of such an insertion or deletion.

In another aspect, the invention provides an isolated nucleic acidmolecule comprising a polynucleotide which hybridizes under stringenthybridization conditions to a portion of the polynucleotide in a nucleicacid molecule of the invention described above. By a polynucleotidewhich hybridizes to a “portion” of a polynucleotide is intended apolynucleotide (either DNA or RNA) hybridizing to at least about 15nucleotides, and more preferably at least about 20 nucleotides, andstill more preferably at least about 30 nucleotides, and even morepreferably more than 30 nucleotides of the reference polynucleotide.These fragments that hybridize to the reference fragments are useful asdiagnostic probes and primers. For the purpose of the invention, twosequences hybridize when they form a double-stranded complex in ahybridization solution of 6×SSC, 0.5% SDS, 5×Denhardt's solution and 100μg of non-specific carrier DNA. See Ausubel et al., section 2.9,supplement 27 (1994). Sequences may hybridize at “moderate stringency,”which is defined as a temperature of 60° C. in a hybridization solutionof 6×SSC, 0.5% SDS, 5×Denhardt's solution and 100 μg of non-specificcarrier DNA. For “high stringency” hybridization, the temperature isincreased to 68° C. Following the moderate stringency hybridizationreaction, the nucleotides are washed in a solution of 2×SSC plus 0.05%SDS for five times at room temperature, with subsequent washes with0.1×SSC plus 0.1% SDS at 60° C. for 1 h. For high stringency, the washtemperature is increased to 68° C. For the purpose of the invention,hybridized nucleotides are those that are detected using 1 ng of aradiolabeled probe having a specific radioactivity of 10,000 cpm/ng,where the hybridized nucleotides are clearly visible following exposureto X-ray film at −70° C. for no more than 72 hours.

The present application is directed to such nucleic acid molecules whichare at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to a nucleic acid sequence described in any of SEQ IDNOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 49-158, 159, 160, 163,168, 173, 178, 183, 188, 193, 198, 203, 208, 215, 220, 225, 230, 247,249, 251, 253, 255, 257, 259, 275-472, 473, 478, 483, 488, 493, 498,503, 513, 515, 517, 519, 521, 533-575, 576, 581, 586, 591, 596, 601,603, 605, 607, 609, 621-767, 768, 773, 778, 783, 788, 793, 795, 797,799, 801, 813-862, 863, 868, 873, 878, 883, 888, 890, 892, 894, 896,908-1040, 1041, 1046, 1051, 1056, 1061, 1071, 1073, 1075, 1077, 1079,1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099, 1101, 1103,1105, 1107, 1109, 1111, 1113, 1161-1571, 1572, 1577, 1582, 1587, 1592,1597, 1602, 1607, 1612, 1617, 1622, 1627, 1632, 1637, 1642, 1647, 1652,1657, 1662, 1667, 1672, 1677, 1682, 1684, 1686, 1688, 1690, 1692, 1694,1696, 1698, 1700, 1702, 1704, 1730-2039, 2040, 2045, 2050, 2055, 2060,2065, 2070, 2075, 2080, 2085, 2090, 2095, 2100, 2102, 2104, 2106, 2108,2120-2338, 2339, 2344, 2349, 2354, 2359, 2364, 2366, 2368, 2370, 2372,2384-2460, 2461, 2466, 2471, 2476 and 2481. Preferred, however, arenucleic acid molecules which are at least 95%, 96%, 97%, 98%, 99% or100% identical to the nucleic acid sequence shown in of SEQ ID NOs: 1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 49-158, 159, 160, 163, 168, 173,178, 183, 188, 193, 198, 203, 208, 215, 220, 225, 230, 247, 249, 251,253, 255, 257, 259, 275-472, 473, 478, 483, 488, 493, 498, 503, 513,515, 517, 519, 521, 533-575, 576, 581, 586, 591, 596, 601, 603, 605,607, 609, 621-767, 768, 773, 778, 783, 788, 793, 795, 797, 799, 801,813-862, 863, 868, 873, 878, 883, 888, 890, 892, 894, 896, 908-1040,1041, 1046, 1051, 1056, 1061, 1071, 1073, 1075, 1077, 1079, 1081, 1083,1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099, 1101, 1103, 1105, 1107,1109, 1111, 1113, 1161-1571, 1572, 1577, 1582, 1587, 1592, 1597, 1602,1607, 1612, 1617, 1622, 1627, 1632, 1637, 1642, 1647, 1652, 1657, 1662,1667, 1672, 1677, 1682, 1684, 1686, 1688, 1690, 1692, 1694, 1696, 1698,1700, 1702, 1704, 1730-2039, 2040, 2045, 2050, 2055, 2060, 2065, 2070,2075, 2080, 2085, 2090, 2095, 2100, 2102, 2104, 2106, 2108, 2120-2338,2339, 2344, 2349, 2354, 2359, 2364, 2366, 2368, 2370, 2372, 2384-2460,2461, 2466, 2471, 2476 and 2481. Differences between two nucleic acidsequences may occur at the 5′ or 3′ terminal positions of the referencenucleotide sequence or anywhere between those terminal positions,interspersed either individually among nucleotides in the referencesequence or in one or more contiguous groups within the referencesequence.

As a practical matter, whether any particular nucleic acid molecule isat least 95%, 96%, 97%, 98% or 99% identical to a reference nucleotidesequence refers to a comparison made between two molecules usingstandard algorithms well known in the art and can be determinedconventionally using publicly available computer programs such as theBLASTN algorithm. See Altschul et al., Nucleic Acids Res. 25:3389-3402(1997).

In one embodiment of the invention, a nucleic acid comprises anantisense strand having about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides,wherein the antisense strand is complementary to a RNA sequence or aportion thereof encoding a protein that controls cell cycle orhomologous recombination, and wherein said siNA further comprises asense strand having about 15 to about 30 (e.g., about 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, andwherein said sense strand and said antisense strand are distinctnucleotide sequences where at least about 15 nucleotides in each strandare complementary to the other strand.

In one embodiment, the present invention provides double-strandednucleic acid molecules of that mediate RNA interference gene silencing.In another embodiment, the siNA molecules of the invention consist ofduplex nucleic acid molecules containing about 15 to about 30 base pairsbetween oligonucleotides comprising about 15 to about 30 (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides. In yet another embodiment, siNA molecules of the inventioncomprise duplex nucleic acid molecules with overhanging ends of about 1to about 32 (e.g., about 1, 2, or 3) nucleotides, for example, about21-nucleotide duplexes with about 19 base pairs and 3′-terminalmononucleotide, dinucleotide, or trinucleotide overhangs. In yet anotherembodiment, siNA molecules of the invention comprise duplex nucleic acidmolecules with blunt ends, where both ends are blunt, or alternatively,where one of the ends is blunt.

An siNA molecule of the present invention may comprise modifiednucleotides while maintaining the ability to mediate RNAi. The modifiednucleotides can be used to improve in vitro or in vivo characteristicssuch as stability, activity, and/or bioavailability. For example, a siNAmolecule of the invention can comprise modified nucleotides as apercentage of the total number of nucleotides present in the siNAmolecule. As such, a siNA molecule of the invention can generallycomprise about 5% to about 100% modified nucleotides (e.g., about 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or 100% modified nucleotides). The actual percentageof modified nucleotides present in a given siNA molecule will depend onthe total number of nucleotides present in the siNA. If the siNAmolecule is single stranded, the percent modification can be based uponthe total number of nucleotides present in the single stranded siNAmolecules. Likewise, if the siNA molecule is double stranded, thepercent modification can be based upon the total number of nucleotidespresent in the sense strand, antisense strand, or both the sense andantisense strands.

VII. Nucleic Acid Constructs

A recombinant nucleic acid vector may, for example, be a linear or aclosed circular plasmid. The vector system may be a single vector orplasmid or two or more vectors or plasmids that together contain thetotal nucleic acid to be introduced into the genome of the bacterialhost. In addition, a bacterial vector may be an expression vector.Nucleic acid molecules as set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 49-158, 159, 160, 161, 162, 163, 168, 173, 178,183, 188, 193, 198, 203, 208, 215, 220, 225, 230, 240-246, 247, 249,251, 253, 255, 257, 259, 275-472, 473, 478, 483, 488, 493, 498, 503,508-512, 513, 515, 517, 519, 521, 533-575, 576, 581, 586, 591, 596, 601,603, 605, 607, 609, 621-767, 768, 773, 778, 783, 788, 793, 795, 797,799, 801, 813-862, 863, 868, 873, 878, 883, 888, 890, 892, 894, 896,908-1040, 1041, 1046, 1051, 1056, 1061, 1066-1070, 1071, 1073, 1075,1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099,1101, 1103, 1105, 1107, 1109, 1111, 1113, 1161-1571, 1572, 1577, 1582,1587, 1592, 1597, 1602, 1607, 1612, 1617, 1622, 1627, 1632, 1637, 1642,1647, 1652, 1657, 1662, 1667, 1672, 1677, 1682, 1684, 1686, 1688, 1690,1692, 1694, 1696, 1698, 1700, 1702, 1704, 1730-2039, 2040, 2045, 2050,2055, 2060, 2065, 2070, 2075, 2080, 2085, 2090, 2095, 2100, 2102, 2104,2106, 2108, 2120-2338, 2339, 2344, 2349, 2354, 2359, 2364, 2366, 2368,2370, 2372, 2384-2460, 2461, 2466, 2471, 2476 and 2481, or fragmentsthereof can, for example, be suitably inserted into a vector under thecontrol of a suitable promoter that functions in one or more microbialhosts to drive expression of a linked coding sequence or other DNAsequence. Many vectors are available for this purpose, and selection ofthe appropriate vector will depend mainly on the size of the nucleicacid to be inserted into the vector and the particular host cell to betransformed with the vector. Each vector contains various componentsdepending on its function (amplification of DNA or expression of DNA)and the particular host cell with which it is compatible. The vectorcomponents for bacterial transformation generally include, but are notlimited to, one or more of the following: a signal sequence, an originof replication, one or more selectable marker genes, and an induciblepromoter allowing the expression of exogenous DNA.

Promoters

“Operably linked”, as used in reference to a regulatory sequence and astructural nucleotide sequence, means that the regulatory sequencecauses regulated expression of the linked structural nucleotidesequence. “Regulatory sequences” or “control elements” refer tonucleotide sequences located upstream (5′ noncoding sequences), within,or downstream (3′ non-translated sequences) of a structural nucleotidesequence, and which influence the timing and level or amount oftranscription, RNA processing or stability, or translation of theassociated structural nucleotide sequence. Regulatory sequences mayinclude promoters, translation leader sequences, introns, enhancers,stem-loop structures, repressor binding sequences, and polyadenylationrecognition sequences and the like.

An expression vector for producing a mRNA can also contain an induciblepromoter that is recognized by the host bacterial organism and isoperably linked to the nucleic acid encoding, for example, the nucleicacid molecule coding the D. v. virgifera mRNA or fragment thereof ofinterest. Inducible promoters suitable for use with bacterial hostsinclude β-lactamase promoter, E. coli λ phage PL and PR promoters, andE. coli galactose promoter, arabinose promoter, alkaline phosphatasepromoter, tryptophan (trp) promoter, and the lactose operon promoter andvariations thereof and hybrid promoters such as the tac promoter.However, other known bacterial inducible promoters are suitable.

In certain embodiments, the genes can be derived from different insectsin order to broaden the range of insects against which the agent iseffective. When multiple genes are targeted for suppression or acombination of expression and suppression, a polycistronic DNA elementcan be fabricated as illustrated and disclosed in Fillatti, ApplicationPublication No. US 2004-0029283.

Selectable Marker Genes

A recombinant DNA vector or construct of the present invention willtypically comprise a selectable marker that confers a selectablephenotype on transformed cells. Selectable markers may also be used toselect for cells that contain the exogenous nucleic acids encodingpolypeptides or proteins of the present invention. The marker may encodebiocide resistance, such as antibiotic resistance (e.g., kanamycin, G418bleomycin, hygromycin, etc.). Examples of selectable markers include,but are not limited to, a neo gene which codes for kanamycin resistanceand can be selected for using kanamycin, G418, etc., a bar gene whichcodes for bialaphos resistance; a nitrilase gene which confersresistance to bromoxynil, and a methotrexate resistant DHFR gene.Examples of such selectable markers are illustrated in U.S. Pat. Nos.5,550,318; 5,633,435; 5,780,708 and 6,118,047.

A recombinant vector or construct of the present invention may alsoinclude a screenable marker. Screenable markers may be used to monitorexpression. Exemplary screenable markers include a β-glucuronidase oruidA gene (GUS) which encodes an enzyme for which various chromogenicsubstrates are known (Jefferson, 1987; Jefferson et al., 1987); aβ-lactamase gene (Sutcliffe et al., 1978), a gene which encodes anenzyme for which various chromogenic substrates are known (e.g., PADAC,a chromogenic cephalosporin); a luciferase gene (Ow et al., 1986) a xylEgene (Zukowsky et al., 1983) which encodes a catechol dioxygenase thatcan convert chromogenic catechols; an α-amylase gene (Ikatu et al.,1990); a tyrosinase gene (Katz et al., 1983) which encodes an enzymecapable of oxidizing tyrosine to DOPA and dopaquinone which in turncondenses to melanin; an α-galactosidase, which catalyzes a chromogenicα-galactose substrate.

A transformation vector can be readily prepared using methods availablein the art. The transformation vector comprises one or more nucleotidesequences that is/are capable of being transcribed to an RNA moleculeand that is/are substantially homologous and/or complementary to one ormore nucleotide sequences encoded by the genome of the insect, such thatupon uptake of the RNA there is down-regulation of expression of atleast one of the respective nucleotide sequences of the genome of thepest.

VIII. Methods for Genetic Engineering

The present invention contemplates introduction of a nucleotide sequenceinto a organism to achieve pest inhibitory levels of expression of oneor more dsRNA molecules. The inventive polynucleotides and polypeptidesmay be introduced into a host cell, such as bacterial or yeast cell, bystandard procedures known in the art for introducing recombinantsequences into a target host cell. Such procedures include, but are notlimited to, transfection, infection, transformation, natural uptake,calcium phosphate, electroporation, microinjection biolistics andmicroorganism-mediated transformation protocols. The methods chosen varywith the host organism.

A transgenic organism of the present invention is one that comprises atleast one cell it its genome in which an exogenous nucleic acid has beenstably integrated. Thus, a transgenic organism may contain onlygenetically modified cells in certain parts of its structure.

Accordingly, the present invention also encompasses a transgenic cell ororganism comprising any of the nucleotide sequences or recombinant DNAconstructs described herein. The invention further encompassesprokaryotic cells (such as, but not limited to, gram-positive andgram-negative bacterial cells) and eukaryotic cells (such as, but notlimited to, yeast cells or plant cells).

For example, the present invention contemplates introducing a targetgene into a bacterium, such as Lactobacillus. The nucleic acidconstructs can be integrated into a bacterial genome with an integratingvector. Integrating vectors typically contain at least one sequencehomologous to the bacterial chromosome that allows the vector tointegrate. Integrations appear to result from recombinations betweenhomologous DNA in the vector and the bacterial chromosome. For example,integrating vectors constructed with DNA from various Bacillus strainsintegrate into the Bacillus chromosome (EP 0 127,328). Integratingvectors may also be comprised of bacteriophage or transposon sequences.Suicide vectors are also known in the art.

Construction of suitable vectors containing one or more of theabove-listed components employs standard recombinant DNA techniques.Isolated plasmids or DNA fragments are cleaved, tailored, and re-ligatedin the form desired to generate the plasmids required. Examples ofavailable bacterial expression vectors include, but are not limited to,the multifunctional E. coli cloning and expression vectors such asBluescript™ (Stratagene, La Jolla, Calif.), in which, for example, a D.v. virgifera protein or fragment thereof, may be ligated into the vectorin frame with sequences for the amino-terminal Met and the subsequent 7residues of β-galactosidase so that a hybrid protein is produced; pINvectors (Van Heeke and Schuster, 1989); and the like.

The invention also contemplates introducing a target gene into a yeastcell. A yeast recombinant construct can typically include one or more ofthe following: a promoter sequence, fusion partner sequence, leadersequence, transcription termination sequence, a selectable marker. Theseelements can be combined into an expression cassette, which may bemaintained in a replicon, such as an extrachromosomal element (e.g.,plasmids) capable of stable maintenance in a host, such as yeast orbacteria. The replicon may have two replication systems, thus allowingit to be maintained, for example, in yeast for expression and in aprokaryotic host for cloning and amplification. Examples of suchyeast-bacteria shuttle vectors include YEp24 (Botstein et al., 1979),pCl/1 (Brake et al., 1984), and YR17 (Stinchcomb et al., 1982). Inaddition, a replicon may be either a high or low copy number plasmid. Ahigh copy number plasmid will generally have a copy number ranging fromabout 5 to about 200, and typically about 10 to about 150. A hostcontaining a high copy number plasmid will preferably have at leastabout 10, and more preferably at least about 20.

Useful yeast promoter sequences can be derived from genes encodingenzymes in the metabolic pathway. Examples of such genes include alcoholdehydrogenase (ADH) (EP 0 284044), enolase, glucokinase,glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase(GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglyceratemutase, and pyruvate kinase (PyK) (EP 0 3215447). The yeast PHO5 gene,encoding acid phosphatase, also provides useful promoter sequences(Myanohara et al., 1983). In addition, synthetic promoters that do notoccur in nature also function as yeast promoters. Examples of suchhybrid promoters include the ADH regulatory sequence linked to the GAPtranscription activation region (U.S. Pat. Nos. 4,876,197 and4,880,734). Examples of transcription terminator sequences and otheryeast-recognized termination sequences, such as those coding forglycolytic enzymes, are known to those of skill in the art.

Alternatively, the expression constructs can be integrated into theyeast genome with an integrating vector. Integrating vectors typicallycontain at least one sequence homologous to a yeast chromosome thatallows the vector to integrate, and preferably contain two homologoussequences flanking the expression construct. Integrations appear toresult from recombinations between homologous DNA in the vector and theyeast chromosome (Orr-Weaver et al., 1983). An integrating vector may bedirected to a specific locus in yeast by selecting the appropriatehomologous sequence for inclusion in the vector. See Orr-Weaver et al.,supra. One or more expression constructs may integrate, possiblyaffecting levels of recombinant protein produced (Rine et al., 1983).

IX. Quantifying Inhibition of Target Gene Expression

Inhibition of target gene expression may be quantified by measuringeither the endogenous target RNA or the protein produced by translationof the target RNA and the consequences of inhibition can be confirmed byexamination of the outward properties of the cell or organism.Techniques for quantifying RNA and proteins are well known to one ofordinary skill in the art. Multiple selectable markers are availablethat confer resistance to ampicillin, bleomycin, chloramphenicol,gentamycin, hygromycin, kanamycin, lincomycin, methotrexate,phosphinothricin, puromycin, spectinomycin, rifampicin, and tetracyclin,and the like.

In certain embodiments gene expression is inhibited by at least 10%,preferably by at least 33%, more preferably by at least 50%, and yetmore preferably by at least 80%. In particularly preferred embodimentsof the invention gene expression is inhibited by at least 80%, morepreferably by at least 90%, more preferably by at least 95%, or by atleast 99% within cells in the pest so a significant inhibition takesplace. Significant inhibition is intended to refer to sufficientinhibition that results in a detectable phenotype (e.g., cessation oflarval growth, paralysis or mortality, etc.) or a detectable decrease inRNA and/or protein corresponding to the target gene being inhibited.Although in certain embodiments of the invention inhibition occurs insubstantially all cells of the pest, in other preferred embodimentsinhibition occurs in only a subset of cells expressing the gene. Forexample, if the target gene plays an essential role in cells in aninsect alimentary tract, inhibition of the gene within these cells issufficient to exert a deleterious effect on the insect.

X. Exposing Pest to dsRNA

A pest can be exposed to a dsRNA in any suitable manner that permitsadministering the dsRNA to the pest. For example, the pest can becontacted with the dsRNA in pure or substantially pure form, for examplean aqueous solution containing the dsRNA. In one embodiment, the insectmay be simply “soaked” or “sprayed” with an aqueous solution comprisingthe dsRNA. Alternatively, the pest may be “sprayed” with a solutioncomprising a dsRNA.

Alternatively, the dsRNA may be linked to a food component of the pest,such as a food component for a mammalian pathogenic pest, in order toincrease uptake of the dsRNA by the insect. Ingestion by a pest permitsdelivery of the pest control agents to the pest and results indown-regulation of a target gene in the host. Methods for oralintroduction may include, for example, directly mixing dsRNA with a,pest's food, as well as engineered approaches in which a species that isused as food is engineered to express the dsRNA or siRNA, then fed tothe pest to be affected. For example, a bacteria, such as Lactobacillus,may be transformed with a target sequence and then fed to a pest. In oneembodiment, for example, the dsRNA or siRNA molecules may beincorporated into, or overlaid on the top of, the insect's diet.

In other embodiments the pest may be contacted with a compositioncontaining the inventive dsRNA. The composition may, in addition to thedsRNA, contain further excipients, diluents, or carriers.

The dsRNA may also be incorporated in the medium in which the pest growsor infests. For example, a dsRNA may be incorporated into a foodcontainer or protective wrapping as a means for inhibiting pestinfestation. Wood, for example, may be treated with a solutioncomprising a dsRNA to prevent pest infestation.

In other embodiments, the dsRNA is expressed in a bacterial or fungalcell and the bacterial or fungal cell is taken up or eaten by the insectspecies.

As illustrated in the examples, bacteria can be engineered to produceany of the dsRNA or dsRNA constructs of the invention. These bacteriacan be eaten by the insect species. When taken up, the dsRNA caninitiate an RNAi response, leading to the degradation of the target mRNAand weakening or killing of the feeding insect. Alternatively, dsRNAproducing bacteria or yeast cells can be sprayed directly onto thecrops.

Some bacteria have a very close interaction with the host plant, suchas, but not limited to, symbiotic Rhizobium with the Legminosea (forexample Soy). Such recombinant bacteria could be mixed with the seeds(for instance as a coating) and used as soil improvers.

A virus such as a baculovirus which specifically infects insects may bealso be used. This ensures safety for mammals, especially humans, sincethe virus will not infect the mammal, so no unwanted RNAi effect willoccur.

Possible applications include intensive greenhouse cultures, forinstance crops that are less interesting from a GMO point of view, aswell as broader field crops such as soy.

This approach has several advantages, eg: since the problem of possibledicing by a plant host is not present, it allows the delivery of largedsRNA fragments into the gut lumen of the feeding pest; the use ofbacteria as insecticides does not involve the generation of transgeniccrops, especially for certain crops where transgenic variants aredifficult to obtain; there is a broad and flexible application in thatdifferent crops can be simultaneously treated on the same field and/ordifferent pests can be simultaneously targeted, for instance bycombining different bacteria producing distinct dsRNAs.

XI. Products

The present invention provides numerous products that can encompass adsRNA for use in controlling pests. For example, the invention providespharmaceutical or veterinary compositions for treating or preventing apest disease or infection of humans or animals, respectively. Suchcompositions comprise at least one dsRNA or RNA construct, or nucleotidesequence or recombinant DNA construct encoding the dsRNA or RNAconstruct, wherein the RNA comprises annealed complementary strands, oneof which has a nucleotide sequence which corresponds to a targetnucleotide sequence of an pest target gene that causes the disease orinfection, and at least one carrier, excipient, or diluent suitable forpharmaceutical use.

Alternatively, a pharmaceutical or veterinary composition may be used asa composition suitable for topical use, such as application on the skinof an animal or human. For example, a dsRNA may be used in a liquidcomposition to be applied to the skin as drops, gel, aerosol, cream,ointment, etc. Additionally, a dsRNA may be integrated into atransdermal patch or other medical device for treating or preventing adisease or condition. Other conventional pharmaceutical dosage forms mayalso be produced, including tablets, capsules, pessaries, transdermalpatches, suppositories, etc. The chosen form will depend upon the natureof the target pest and hence the nature of the disease it is desired totreat.

Oral vaccines, for example, can be produced using the inventiveconstructs and methods. For example, a vaccine can be constructed byproducing a dsRNA in bacteria (e.g. lactobacillus) which can be includedin food and functions as an oral vaccine against insect infection.Accordingly, the invention provides constructs and methods for treatingand/or preventing a pest disease or condition, comprising administeringto a subject in need of such treatment and/or prevention, any of thecompositions as herein described, said composition comprising at leastone double-stranded RNA or double stranded RNA construct comprisingannealed complementary strands, one of which has a nucleotide sequencewhich is complementary to at least part of a nucleotide sequence of apest target gene that causes the disease or condition.

While the inventive compositions may be used for treating a disease orcondition in a subject patient, the compositions and methods may also beused as a means for protecting a substrate or material from pestinfestation. The nature of the excipients included in the compositionand the physical form of the composition may vary depending upon thenature of the substrate that it is desired to treat.

For example, such a composition may be a coating or a powder that can beapplied to a substrate as a means for protecting the substrate frominfestation by an insect and thereby preventing pest-induced damage tothe substrate or material. Thus, in one embodiment, the composition isin the form of a coating on a suitable surface which adheres to, and iseventually ingested by an insect which comes into contact with thecoating. Such a composition can be used to protect any substrate ormaterial that is susceptible to infestation by or damage caused by apest, for example foodstuffs and other perishable materials, andsubstrates such as wood.

For example, the composition may be a liquid that is brushed or sprayedonto or imprinted into the material or substrate to be treated. Thus, ahuman user can spray the insect or the substrate directly with thecomposition

For example, houses and other wood products can be destroyed bytermites, powder post beetles, and carpenter ants. By treating wood orhouse siding with a composition comprising a dsRNA, it may be possibleto reduce pest infestation. Likewise, a tree trunk may be treated with acomposition comprising a dsRNA.

Flour beetles, grain weevils, meal moths, and other pests feed on storedgrain, cereals, pet food, powdered chocolate, and almost everything elsein the kitchen pantry that is not protected. Accordingly, the presentinvention provides a means for treating cereal boxes and other foodstorage containers and wrapping with a composition comprising a targetdsRNA.

Larvae of clothes moths eat clothes made from animal products, such asfur, silk and wool. Thus, it may be desirable to treat hangers, closetorganizers, and garment bags with the inventive dsRNA. Book lice andsilverfish are pests of libraries because they eat the starchy glue inthe bindings of books. Accordingly, the present invention providescompositions for treating books from pest infestation and destruction.

In one embodiment, the composition is in the form of a bait. The bait isdesigned to lure the insect to come into contact with the composition.Upon coming into contact therewith, the composition is then internalizedby the insect, by ingestion for example and mediates RNAi to thus killthe insect. The bait may depend on the species being targeted. Anattractant may also be used. The attractant may be a pheromone, such asa male or female pheromone for example. The attractant acts to lure theinsect to the bait, and may be targeted for a particular insect or mayattract a whole range of insects. The bait may be in any suitable form,such as a solid, paste, pellet or powdered form.

The bait may also be carried away by the insect back to the colony. Thebait may then act as a food source for other members of the colony, thusproviding an effective control of a large number of insects andpotentially an entire insect pest colony. This is an advantageassociated with use of the double stranded RNA or bacteria expressingthe dsRNA of the invention, because the delayed action of the RNAimediated effects on the pests allows the bait to be carried back to thecolony, thus delivering maximal impact in terms of exposure to theinsects.

The baits may be provided in a suitable “housing” or “trap”. Suchhousings and traps are commercially available and existing traps may beadapted to include the compositions of the invention. The housing ortrap may be box-shaped for example, and may be provided in pre-formedcondition or may be formed of foldable cardboard for example. Suitablematerials for a housing or trap include plastics and cardboard,particularly corrugated cardboard. The inside surfaces of the traps maybe lined with a sticky substance in order to restrict movement of theinsect once inside the trap. The housing or trap may contain a suitabletrough inside which can hold the bait in place. A trap is distinguishedfrom a housing because the insect can not readily leave a trap followingentry, whereas a housing acts as a “feeding station” which provides theinsect arachnid with a preferred environment in which they can feed andfeel safe from predators.

It is clear that numerous products and substrates can be treated withthe inventive compositions for reducing pest infestation. Of course, thenature of the excipients and the physical form of the composition mayvary depending upon the nature of the substrate that is desired totreat. For example, the composition may be a liquid that is brushed orsprayed onto or imprinted into the material or substrate to be treated,or a coating that is applied to the material or substrate to be treated.

Specific examples are presented below of methods for identifying targetsequences and introducing the sequences into various cells andcompositions. They are meant to be exemplary and not as limitations onthe present invention.

Example 1 Silencing C. elegans Target Genes in C. elegans in HighThroughput Screening

A C. elegans genome wide library was prepared in the pGN9A vector (WO01/88121) between two identical T7-promoters and terminators, drivingits expression in the sense and antisense direction upon expression ofthe T7 polymerase, which was induced by IPTG.

This library was transformed into the bacterial strain AB301-105 (DE3)in 96 well plate format. For the genome wide screening, these bacterialcells were fed to the nuclease deficient C. elegans nuc-1 (e1392)strain.

Feeding the dsRNA produced in the bacterial strain AB301-105 (DE3), toC. elegans nuc-1 (e1392) worms, was performed in a 96 well plate formatas follows: nuc-1 eggs were transferred to a separate plate and allowedto hatch simultaneously at 20° C. for synchronization of the L1generation. 96 well plates were filled with 100 μL liquid growth mediumcomprising IPTG and with 10 μL bacterial cell culture of OD₆₀₀1AB301-105 (DE3) of the C. elegans dsRNA library carrying each a vectorwith a C. elegans genomic fragment for expression of the dsRNA. To eachwell, 4 of the synchronized L1 worms were added and were incubated at25° C. for at least 4 to 5 days. These experiments were performed inquadruplicate. In the screen 6 controls were used:

-   -   pGN29=negative control, wild type    -   pGZ1=unc-22=twitcher phenotype    -   pGZ18=chitin synthase=embryonic lethal    -   pGZ25=pos-1=embryonic lethal    -   pGZ59=bli-4D=acute lethal    -   ACC=acetyl co-enzyme A carboxylase=acute lethal

After 5 days, the phenotype of the C. elegans nuc-1 (e1392) worms fedwith the bacteria producing dsRNA were compared to the phenotype ofworms fed with the empty vector (pGN29) and the other controls. Theworms that were fed with the dsRNA were screened for lethality (acute orlarval) lethality for the parent (Po) generation, (embryonic) lethalityfor the first filial (F1) generation, or for growth retardation of Po asfollows: (i) Acute lethality of Po: L1's have not developed and aredead, this phenotype never gives progeny and the well looks quite empty;(ii) (Larval) lethality of Po: Po died in a later stage than L1, thisphenotype also never gives progeny. Dead larvae or dead adult worms arefound in the wells; (iii) Lethality for F1: L1's have developed untiladult stage and are still alive. This phenotype has no progeny. This canbe due to sterility, embryonic lethality (dead eggs on the bottom ofwell), embryonic arrest or larval arrest (eventually ends up beinglethal): (iv) Arrested in growth and growth retardation/delay: Comparedto a well with normal development and normal # of progeny.

For the target sequences presented in Table 1A, it was concluded thatdsRNA mediated silencing of the C. elegans target gene in nematodes,such as C. elegans, had a fatal effect on the growth and viability ofthe worm.

Subsequent to the above dsRNA silencing experiment, a more detailedphenotyping experiment was conducted in C. elegans in a high throughputformat on 24 well plates. The dsRNA library produced in bacterial strainAB301-105 (DE3), as described above, was fed to C. elegans nuc-1 (e1392)worms on 24 well plates as follows: nuc-1 eggs were transferred to aseparate plate and allowed to hatch simultaneously at 20° C. forsynchronization of the L1 generation. Subsequently 100 of thesynchronized L1 worms were soaked in a mixture of 500 μL S-complete fedmedium, comprising 5 μg/mL cholesterol, 4 μL/mL PEG and 1 mM IPTG, and500 μL of bacterial cell culture of OD₆₀₀1 AB301-105 (DE3) of the C.elegans dsRNA library carrying each a vector with a C. elegans genomicfragment for expression of the dsRNA. The soaked L1 worms were rolledfor 2 hours at 25° C.

After centrifugation and removal of 950 μL of the supernatant, 5 μL ofthe remaining and resuspended pellet (comprising about 10 to 15 worms)was transferred in the middle of each well of a 24 well plate, filledwith a layer of agar LB broth. The inoculated plate was incubated at 25°C. for 2 days. At the adult stage, 1 adult worm was singled andincubated at 25° C. for 2 days for inspection of its progeny. The otheradult worms are inspected in situ on the original 24 well plate. Theseexperiments were performed in quadruplicate.

This detailed phenotypic screen was repeated with a second batch ofworms, the only difference being that the worms of the second batch wereincubated at 20° C. for 3 days.

The phenotype of the worms fed with C. elegans dsRNA was compared to thephenotype of C. elegans nuc-1 (e1392) worms fed with the empty vector.

Based on this experiment, it was concluded that silencing the C. eleganstarget genes as represented in Table 1A had a fatal effect on the growthand viability of the worm and that the target gene is essential to theviability of nematodes. Therefore these genes are good target genes tocontrol (kill or prevent from growing) nematodes via dsRNA mediated genesilencing. Accordingly, the present invention encompasses the use ofnematode orthologs of the above C. elegans target gene to controlnematode infestation in a variety of organisms and materials.

Example 2 Identification of D. melanogaster Orthologs

As described above in Example 1, numerous C. elegans lethal sequenceswere identified and can be used for identifying orthologs in otherspecies and genera. For example, the C. elegans lethal sequences can beused to identify orthologous D. melanogasters sequences. That is, eachC. elegans sequence can be queried against a public database, such asGenBank, for orthologous sequences in D. melanogaster. Potential D.melanogaster orthologs were selected that share a high degree ofsequence homology (E value preferably less than or equal to 1E-30) andthe sequences are blast reciprocal best hits, the latter means that thesequences from different organisms (e.g. C. elegans and D. melanogaster)are each other's top blast hits. For example, sequence C from C. elegansis compared against sequences in D. melanogaster using BLAST. Ifsequence C has the D. melanogaster sequence D as best hit and when D iscompared to all the sequences of C. elegans, also turns out to besequence C, then D and C are reciprocal best hits. This criterium isoften used to define orthology, meaning similar sequences of differentspecies, having similar function. The D. melanogaster sequenceidentifiers are represented in Table 1A.

Example 3 Leptinotarsa decemlineata (Colorado Potato Beetle) A. CloningPartial Gene Sequences from Leptinotarsa decemlineata

High quality, intact RNA was isolated from 4 different larval stages ofLeptinotarsa decemlineata (Colorado potato beetle; source: Jeroen vanSchaik, Entocare CV Biologische Gewasbescherming, Postbus 162, 6700 ADWageningen, the Netherlands) using TRIzol Reagent (Cat. Nr.15596-026/15596-018, Invitrogen, Rockville, Md., USA) following themanufacturer's instructions. Genomic DNA present in the RNA preparationwas removed by DNase treatment following the manufacturer's instructions(Cat. Nr. 1700, Promega). cDNA was generated using a commerciallyavailable kit (SuperScript™ III Reverse Transcriptase, Cat. Nr.18080044, Invitrogen, Rockville, Md., USA) following the manufacturer'sinstructions.

To isolate cDNA sequences comprising a portion of the LD001, LD002,LD003, LD006, LD007, LD010, LD011, LD014, LD015, LD016 and LD018 genes,a series of PCR reactions with degenerate primers were performed usingAmplitaq Gold (Cat. Nr. N8080240, Applied Biosystems) following themanufacturer's instructions.

The sequences of the degenerate primers used for amplification of eachof the genes are given in Table 2-LD, which displays Leptintarsadecemlineata target genes including primer sequences and cDNA sequencesobtained. These primers were used in respective PCR reactions with thefollowing conditions: 10 minutes at 95° C., followed by 40 cycles of 30seconds at 95° C., 1 minute at 55° C. and 1 minute at 72° C., followedby 10 minutes at 72° C. The resulting PCR fragments were analyzed onagarose gel, purified (QIAquick Gel Extraction kit, Cat. Nr. 28706,Qiagen), cloned into the pCR8/GW/topo vector (Cat. Nr. K2500 20,Invitrogen), and sequenced. The sequences of the resulting PCR productsare represented by the respective SEQ ID NOs as given in Table 2-LD andare referred to as the partial sequences. The corresponding partialamino acid sequence are represented by the respective SEQ ID NOs asgiven in Table 3-LD, where the start of the reading frame is indicatedin brackets.

B. dsRNA Production of the Leptinotarsa decemlineata Genes

dsRNA was synthesized in milligram amounts using the commerciallyavailable kit T7 Ribomax™ Express RNAi System (Cat. Nr. P1700, Promega).First two separate single 5′ T7 RNA polymerase promoter templates weregenerated in two separate PCR reactions, each reaction containing thetarget sequence in a different orientation relative to the T7 promoter.

For each of the target genes, the sense T7 template was generated usingspecific T7 forward and specific reverse primers. The sequences of therespective primers for amplifying the sense template for each of thetarget genes are given in Table 8-LD. The conditions in the PCRreactions were as follows: 4 minutes at 95° C., followed by 35 cycles of30 seconds at 95° C., 30 seconds at 55° C. and 1 minute at 72° C.,followed by 10 minutes at 72° C. The anti-sense T7 template wasgenerated using specific forward and specific T7 reverse primers in aPCR reaction with the same conditions as described above. The sequencesof the respective primers for amplifying the anti-sense template foreach of the target genes are given in Table 8-LD. The resulting PCRproducts were analyzed on agarose gel and purified by PCR purificationkit (Qiaquick PCR Purification Kit, Cat. Nr. 28106, Qiagen) and NaClO₄precipitation. The generated T7 forward and reverse templates were mixedto be transcribed and the resulting RNA strands were annealed, DNase andRNase treated, and purified by sodium acetate, following themanufacturer's instructions. The sense strand of the resulting dsRNA foreach of the target genes is given in Table 8-LD. Table 8-LD displayssequences for preparing ds RNA fragments of Leptinotarsa decemlineatatarget sequences and concatemer sequences, including primer sequences.

C. Screening dsRNA Targets Using Artificial Diet for Activity AgainstLeptinotarsa decemlineata

Artificial diet for the Colorado potato beetle was prepared as follows(adapted from Gelman et al., 2001, J. Ins. Sc., vol. 1, no. 7, 1-10):water and agar were autoclaved, and the remaining ingredients (shown inTable 2 below) were added when the temperature dropped to 55° C. At thistemperature, the ingredients were mixed well before the diet wasaliquoted into 24-well plates (Nunc) with a quantity of 1 ml of diet perwell. The artificial diet was allowed to solidify by cooling at roomtemperature. Diet was stored at 4° C. for up to three weeks.

TABLE 2 Ingredients for Artificial diet Ingredients Volume for 1 L water768 ml agar 14 g rolled oats 40 g Torula yeast 60 g lactalbumin 30 ghydrolysate casein 10 g fructose 20 g Wesson salt mixture 4 g tomatofruit powder 12.5 g potato leaf powder 25 g b-sitosterol 1 g sorbic acid0.8 g methyl paraben 0.8 g Vanderzant vitamin 12 g mix neomycin sulfate0.2 g aureomycin 0.130 g rifampicin 0.130 g chloramphenicol 0.130 gnystatin 0.050 g soybean oil 2 ml wheat germ oil 2 ml

Fifty μl of a solution of dsRNA at a concentration of 1 mg/ml wasapplied topically onto the solid artificial diet in the wells of themultiwell plate. The diet was dried in a laminair flow cabin. Pertreatment, twenty-four Colorado potato beetle larvae (2^(nd) stage),with two insects per well, were tested. The plates were stored in theinsect rearing chamber at 25±2° C., 60% relative humidity, with a 16:8hours light:dark photoperiod. The beetles were assessed as live or deadevery 1, 2 or 3 days. After seven days, for targets LD006, LD007, LD010,LD011, and LD014, the diet was replaced with fresh diet with topicallyapplied dsRNA at the same concentration (1 mg/ml); for targets LD001,LD002, LD003, LD015, and LD016, the diet was replaced with fresh dietonly. The dsRNA targets were compared to diet only or diet withtopically applied dsRNA corresponding to a fragment of the GFP (greenfluorescent protein) coding sequence (SEQ ID NO: 235).

Feeding artificial diet containing intact naked dsRNAs to L.decemlineata larvae resulted in significant increases in larvalmortalities as indicated in two separate bioassays (FIGS. 1LD-2LD).

All dsRNAs tested resulted ultimately in 100% mortality after 7 to 14days. Diet with or without GFP dsRNA sustained the insects throughoutthe bioassays with very little or no mortality.

Typically, in all assays observed, CPB second-stage larvae fed normallyon diet with or without dsRNA for 2 days and molted to the third larvalstage. At this new larval stage the CPB were observed to reducesignificantly or stop altogether their feeding, with an increase inmortality as a result.

D. Bioassay of dsRNA Targets Using Potato Leaf Discs for ActivityAgainst the Leptinotarsa decemlineata

An alternative bioassay method was employed using potato leaf materialrather than artificial diet as food source for CPB. Discs ofapproximately 1.1 cm in diameter (or 0.95 cm²) were cut out off leavesof 2 to 3-week old potato plants using a suitably-sized cork borer.Treated leaf discs were prepared by applying 20 μl of a 10 ng/μlsolution of target LD002 dsRNA or control gfp dsRNA on the adaxial leafsurface. The leaf discs were allowed to dry and placed individually in24 wells of a 24-well multiplate (Nunc). A single second-larval stageCPB was placed into each well, which was then covered with tissue paperand a multiwell plastic lid. The plate containing the insects and leafdiscs were kept in an insect chamber at 28° C. with a photoperiod of 16h light/8 h dark. The insects were allowed to feed on the leaf discs for2 days after which the insects were transferred to a new platecontaining fresh treated leaf discs. Thereafter, the insects weretransferred to a plate containing untreated leaf discs every day untilday 7. Insect mortality and weight scores were recorded.

Feeding potato leaf discs with surface-applied intact naked dsRNA oftarget LD002 to L. decemlineata larvae resulted in a significantincrease in larval mortalities (i.e. at day 7 all insects were dead;100% mortality) whereas control gfp dsRNA had no effect on CPB survival.Target LD002 dsRNA severely affected the growth of the larvae after 2 to3 days whereas the larvae fed with gfp dsRNA at the same concentrationdeveloped as normal (FIG. 3-LD).

D. Screening Shorter Versions of dsRNAs Using Artificial Diet forActivity Against Leptinotarsa decemlineata

This example exemplifies the finding that shorter (60 or 100 bp) dsRNAfragments on their own or as concatemer constructs are sufficient incausing toxicity towards the Colorado potato beetle.

LD014, a target known to induce lethality in Colorado potato beetle, wasselected for this example. This gene encodes a V-ATPase subunit E (SEQID NO: 15).

A 100 base pair fragment, LD014_F1, at position 195-294 on SEQ ID NO: 15(SEQ ID NO: 159) and a 60 base pair fragment, LD014_F2, at position235-294 on SEQ ID NO: (SEQ ID NO: 160) were further selected. See alsoTable 7-LD.

Two concatemers of 300 base pairs, LD014_C1 and LD014_C2, were designed(SEQ ID NO: 161 and SEQ ID NO: 162). LD014_C1 contained 3 repeats of the100 base pair fragment described above (SEQ ID NO: 159) and LD014_C2contained 5 repeats of the 60 base pair fragment described above (SEQ IDNO: 160). See also Table 7-LD.

The fragments LD014_F1 and LD014_F2 were synthesized as sense andantisense primers. These primers were annealed to create the doublestrands DNA molecules prior to cloning. XbaI and XmaI restrictions siteswere included at the 5′ and 3′ ends of the primers, respectively, tofacilitate the cloning.

The concatemers were made as 300 base pairs synthetic genes. XbaI andXmaI restrictions sites were included at the 5′ and 3′ ends of thesynthetic DNA fragments, respectively, to facilitate the cloning.

The 4 DNA molecules, i.e. the 2 single units (LD014_F1 & LD014_F2) andthe 2 concatemers (LD014_C1 & LD014_C2), were digested with XbaI andXmaI and subcloned in pBluescriptII SK+ linearised by XbaI and XmaIdigests, resulting in recombinant plasmids p1, p2, p3, & p4,respectively.

Double-stranded RNA production: dsRNA was synthesized using thecommercially available kit T7 Ribomax™ Express RNAi System (Cat. Nr.P1700, Promega). First two separate single 5′ T7 RNA polymerase promotertemplates were generated in two separate PCR reactions, each reactioncontaining the target sequence in a different orientation relative tothe T7 promoter. For LD014_F1, the sense T7 template was generated usingthe specific T7 forward primer oGBM159 and the specific reverse primeroGBM164 (represented herein as SEQ ID NO: 204 and SEQ ID NO: 205,respectively) in a PCR reaction with the following conditions: 4 minutesat 95° C., followed by 35 cycles of 30 seconds at 95° C., 30 seconds at55° C. and 1 minute at 72° C., followed by 10 minutes at 72° C. Theanti-sense T7 template was generated using the specific forward primeroGBM163 and the specific T7 reverse primer oGBM160 (represented hereinas SEQ ID NO: 206 and SEQ ID NO: 207, respectively) in a PCR reactionwith the same conditions as described above. The resulting PCR productswere analyzed on agarose gel and purified by PCR purification kit(Qiaquick PCR Purification Kit, Cat. Nr. 28106, Qiagen) and NaClO₄precipitation. The generated T7 forward and reverse templates were mixedto be transcribed and the resulting RNA strands were annealed, Dnase andRnase treated, and purified by sodium acetate, following themanufacturer's instructions. The sense strand of the resulting dsRNA isherein represented by SEQ ID NO: 203.

For LD014_F2, the sense T7 template was generated using the specific T7forward primer oGBM161 and the specific reverse primer oGBM166(represented herein as SEQ ID NO: 209 and SEQ ID NO: 210, respectively)in a PCR reaction with the following conditions: 4 minutes at 95° C.,followed by 35 cycles of 30 seconds at 95° C., 30 seconds at 55° C. and1 minute at 72° C., followed by 10 minutes at 72° C. The anti-sense T7template was generated using the specific forward primer oGBM165 and thespecific T7 reverse primer oGBM162 (represented herein as SEQ ID NO: 211and SEQ ID NO: 212, respectively) in a PCR reaction with the sameconditions as described above. The resulting PCR products were analyzedon agarose gel and purified by PCR purification kit (Qiaquick PCRPurification Kit, Cat. Nr. 28106, Qiagen) and NaClO₄ precipitation. Thegenerated T7 forward and reverse templates were mixed to be transcribedand the resulting RNA strands were annealed, Dnase and Rnase treated,and purified by sodium acetate, following the manufacturer'sinstructions. The sense strand of the resulting dsRNA is hereinrepresented by SEQ ID NO: 208.

Also for the concatemers, separate single 5′ T7 RNA polymerase promotertemplates were generated in two separate PCR reactions, each reactioncontaining the target sequence in a different orientation relative tothe T7 promoter. The recombinant plasmids p3 and p4 containing LD014_C1& LD014_C2 were linearised with XbaI or XmaI, the two linear fragmentsfor each construct purified and used as template for the in vitrotranscription assay, using the T7 promoters flanking the cloning sites.Double-stranded RNA was prepared by in vitro transcription using the T7RiboMAX™ Express RNAi System (Promega). The sense strands of theresulting dsRNA for LD014_C1 and LD014_C2 are herein represented by SEQID NO: 213 and 2114, respectively.

Shorter sequences of target LD014 and concatemers were able to inducelethality in Leptinotarsa decemlineata, as shown in FIG. 4-LD.

G. Screening dsRNAs at Different Concentrations Using Artificial Dietfor Activity Against Leptinotarsa decemlineata

Fifty μl of a solution of dsRNA at serial ten-fold concentrations from 1μg/μl (for target LD027 from 0.1 μg/μl) down to 0.01 ng/μl was appliedtopically onto the solid artificial diet in the wells of a 24-well plate(Nunc). The diet was dried in a laminair flow cabin. Per treatment,twenty-four Colorado potato beetle larvae (2^(nd) stage), with twoinsects per well, were tested. The plates were stored in the insectrearing chamber at 25±2° C., 60% relative humidity, with a 16:8 hourslight:dark photoperiod. The beetles were assessed as live or dead atregular intervals up to day 14. After seven days, the diet was replacedwith fresh diet with topically applied dsRNA at the same concentrations.The dsRNA targets were compared to diet only.

Feeding artificial diet containing intact naked dsRNAs of differenttargets to L. decemlineata larvae resulted in high larval mortalities atconcentrations as low as between 0.1 and 10 ng dsRNA/μl as shown in FIG.5-LD.

H. Cloning of a CPB Gene Fragment in a Vector Suitable for BacterialProduction of Insect-Active Double-Stranded RNA

While any efficient bacterial promoter may be used, a DNA fragmentcorresponding to an MLB gene target was cloned in a vector for theexpression of double-stranded RNA in a bacterial host (See WO 00/01846).

The sequences of the specific primers used for the amplification oftarget genes are provided in Table 8. The template used is thepCR8/GW/topo vector containing any of target sequences. The primers areused in a PCR reaction with the following conditions: 5 minutes at 98°C., followed by 30 cycles of 10 seconds at 98° C., 30 seconds at 55° C.and 2 minutes at 72° C., followed by 10 minutes at 72° C. The resultingPCR fragment is analyzed on agarose gel, purified (QIAquick GelExtraction kit, Cat. Nr. 28706, Qiagen), blunt-end cloned into SrfI-linearized pGNA49A vector (reference to WO00188121A1), and sequenced.The sequence of the resulting PCR product corresponds to the respectivesequence as given in Table 8. The recombinant vector harboring thissequence is named pGBNJ003.

The sequences of the specific primers used for the amplification oftarget gene fragment LD010 are provided in Table 8 (forward primer SEQID NO: 191 and reverse primer SEQ ID NO: 190). The template used was thepCR8/GW/topo vector containing the LD010 sequence (SEQ ID NO: 11). Theprimers were used in a PCR reaction with the following conditions: 5minutes at 98° C., followed by 30 cycles of 10 seconds at 98° C., 30seconds at 55° C. and 2 minutes at 72° C., followed by 10 minutes at 72°C. The resulting PCR fragment was analyzed on agarose gel, purified(QIAquick Gel Extraction kit, Cat. Nr. 28706, Qiagen), blunt-end clonedinto Srf I-linearized pGNA49A vector (reference to WO 00/188121A1), andsequenced. The sequence of the resulting PCR product corresponds to SEQID NO: 188 as given in Table 8. The recombinant vector harboring thissequence was named pGBNJ003.

I. Expression and Production of a Double-Stranded RNA Target in TwoStrains of Escherichia coli: (1) AB309-105, and, (2) BL21(DE3)

The procedures described below were followed in order to expresssuitable levels of insect-active double-stranded RNA of target LD010 inbacteria. An RNaseIII-deficient strain, AB309-105, was used incomparison to wild-type RNaseIII-containing bacteria, BL21(DE3).

Transformation of AB309-105 and BL21(DE3)

Three hundred ng of the plasmid was added to and gently mixed in a 50 μlaliquot of ice-chilled chemically competent E. coli strain AB309-105 orBL21(DE3). The cells were incubated on ice for 20 minutes beforesubjecting them to a heat shock treatment of 37° C. for 5 minutes, afterwhich the cells were placed back on ice for a further 5 minutes. Fourhundred and fifty μl of room temperature SOC medium was added to thecells and the suspension incubated on a shaker (250 rpm) at 37° C. for 1hour. One hundred μl of the bacterial cell suspension was transferred toa 500 ml conical flask containing 150 ml of liquid Luria-Bertani (LB)broth supplemented with 100 μg/ml carbenicillin antibiotic. The culturewas incubated on an Innova 4430 shaker (250 rpm) at 37° C. overnight (16to 18 hours).

Chemical Induction of Double-Stranded RNA Expression in AB309-105 andBL21 (DE3)

Expression of double-stranded RNA from the recombinant vector, pGBNJ003,in the bacterial strain AB309-105 or BL21(DE3) was made possible sinceall the genetic components for controlled expression are present. In thepresence of the chemical inducer isopropylthiogalactoside, or IPTG, theT7 polymerase will drive the transcription of the target sequence inboth antisense and sense directions since these are flanked byoppositely oriented T7 promoters.

The optical density at 600 nm of the overnight bacterial culture wasmeasured using an appropriate spectrophotometer and adjusted to a valueof 1 by the addition of fresh LB broth. Fifty ml of this culture wastransferred to a 50 ml Falcon tube and the culture then centrifuged at3000 g at 15° C. for 10 minutes. The supernatant was removed and thebacterial pellet resuspended in 50 ml of fresh S complete medium (SNCmedium plus 5 μg/ml cholesterol) supplemented with 100 μg/mlcarbenicillin and 1 mM IPTG. The bacteria were induced for 2 to 4 hoursat room temperature.

Heat Treatment of Bacteria

Bacteria were killed by heat treatment in order to minimize the risk ofcontamination of the artificial diet in the test plates. However, heattreatment of bacteria expressing double-stranded RNA is not aprerequisite for inducing toxicity towards the insects due to RNAinterference. The induced bacterial culture was centrifuged at 3000 g atroom temperature for 10 minutes, the supernatant discarded and thepellet subjected to 80° C. for 20 minutes in a water bath. After heattreatment, the bacterial pellet was resuspended in 1.5 ml MilliQ waterand the suspension transferred to a microfuge tube. Several tubes wereprepared and used in the bioassays for each refreshment. The tubes werestored at −20° C. until further use.

J. Laboratory Trials to Test Escherichia coli Expressing dsRNA TargetLD010 Against Leptinotarsa decemlineata

Two bioassay methods were employed to test double-stranded RNA producedin Escherichia coli against larvae of the Colorado potato beetle: (1)artificial diet-based bioassay, and, (2) plant-based bioassay.

Artificial Diet-Based Bioassays

Artificial diet for the Colorado potato beetle was prepared as describedpreviously in Example 4. A half milliliter of diet was dispensed intoeach of the wells of a 48-well multiwell test plate (Nunc). For everytreatment, fifty μl of an OD 1 suspension of heat-treated bacteria(which is equivalent to approximately 5×10⁷ bacterial cells) expressingdsRNA was applied topically onto the solid diet in the wells and theplates were allowed to dry in a laminair flow cabin. Per treatment,forty-eight 2^(nd) stage Colorado potato beetle larvae, one in each wellcontaining diet and bacteria, were tested. Each row of a plate (i.e. 8wells) was considered as one replicate. The plates were kept in theinsect rearing chamber at 25±2° C., 60±5% relative humidity, with a 16:8hours light:dark photoperiod. After every 4 days, the beetles weretransferred to fresh diet containing topically-applied bacteria. Thebeetles were assessed as alive or dead every one or three days postinfestation. For the survivors, growth and development in terms oflarval weight was recorded on day 7 post infestation.

For RNaseIII-deficient E. coli strain AB309-105, bacteria containingplasmid pGBNJO03 and those containing the empty vector pGN29 (referenceto WO 00/188121A1) were tested in bioassays for CPB toxicity. Bacteriaharboring the pGBNJO03 plasmid showed a clear increase in insectmortality with time, whereas little or no mortality was observed forpGN29 and diet only control (FIGS. 6 a-LD & 7 a-LD). The growth anddevelopment of Colorado potato beetle larval survivors, 7 days afterfeeding on artificial diet containing bacteria expressing dsRNA targetLD010, was severely impeded (Table 10-LD, FIG. 8 a-LD).

For E. coli strain BL21(DE3), bacteria containing plasmid pGBNJ003 andthose containing the empty vector pGN29 were tested against the Coloradopotato beetle larvae. Similar detrimental effects were observed onlarvae fed diet supplemented with BL21(DE3) bacteria as for theRNAseIII-deficient strain, AB309-105 (FIGS. 6 b-LD & 7 b-LD). However,the number of survivors for the five clones were higher for BL21(DE3)than for AB309-105; at day 12, average mortality values wereapproximately 25% lower for this strain compared to the RNase IIIdeficient strain. Also, the average weights of survivors fed on dietcontaining BL21(DE3) expressing dsRNA corresponding to target LD010 wasseverely reduced (Table 10-LD, FIG. 8 b-LD).

The delay in growth and development of the CPB larvae fed on dietcontaining either of the two bacterial strains harboring plasmidpGBNJ003 was directly correlated to feeding inhibition since no frasswas visible in the wells of refreshed plates from day 4 onwards whencompared to bacteria harboring the empty vector pGN29 or the diet onlyplate. This observation was similar to that where CPB was fed on invitro transcribed double-stranded RNA topically applied to artificialdiet (see Example 3D); here, cessation of feeding occurred from day 2onwards on treated diet.

Plant-Based Bioassays

Whole potato plants were sprayed with suspensions of chemically inducedbacteria expressing dsRNA prior to feeding the plants to CPB larvae. Thepotato plants of variety line 5′ were grown from tubers to the 8-12unfolded leaf stage in a plant growth room chamber with the followingconditions: 25±2° C., 60% relative humidity, 16:8 hour light:darkphotoperiod. The plants were caged by placing a 500 ml plastic bottleupside down over the plant with the neck of the bottle firmly placed inthe soil in a pot and the base cut open and covered with a fine nylonmesh to permit aeration, reduce condensation inside and prevent larvalescape. Fifteen Colorado potato beetle larvae at the L1 stage wereplaced on each treated plant in the cage. Plants were treated with asuspension of E. coli AB309-105 harboring the pGBNJ003 plasmids (clone1; FIG. 7 a-LD) or pGN29 plasmid (clone 1; see FIG. 7 a-LD). Differentquantities of bacteria were applied to the plants: 66, 22, and 7 units,where one unit is defined as 109 bacterial cells in 1 ml of a bacterialsuspension at optical density value of 1 at 600 nm wavelength. In eachcase, a total volume of 1.6 ml was sprayed on the plant with the aid ofa vaporizer. One plant was used per treatment in this trial. The numberof survivors were counted and the weight of each survivor recorded.

Spraying plants with a suspension of E. coli bacterial strain AB309-105expressing target dsRNA from pGBNJO03 led to a dramatic increase ininsect mortality when compared to pGN29 control. The mortality count wasmaintained when the amount of bacteria cell suspension was diluted9-fold (FIG. 9-LD). The average weights of the larval survivors at day11 on plants sprayed with bacteria harboring the pGBNJO03 vector wereapproximately 10-fold less than that of pGN29 (FIG. 10-LD). Feedingdamage by CPB larvae of the potato plant sprayed with bacteriacontaining the pGBNJO03 plasmid was much reduced when compared to thedamage incurred on a potato plant sprayed with bacteria containing theempty vector pGN29 (FIG. 11-LD).

These experiments showed that double-stranded RNA corresponding to aninsect gene target sequence produced in either wild-type orRNaseIII-deficient bacterial expression systems is toxic towards theinsect in terms of substantial increases in insect mortality andgrowth/development delay for larval survivors. It is also clear fromthese experiments that an exemplification was provided for the effectiveprotection of plants/crops from insect damage by the use of a spray of aformulation consisting of bacteria expressing double-stranded RNAcorresponding to an insect gene target.

K. Testing Various Culture Suspension Densities of Escherichia coliExpressing dsRNA Target LD010 Against Leptinotarsa decemlineata

Preparation and treatment of bacterial cultures are described in Example3J. Three-fold serial dilutions of cultures (starting from 0.25 unitequivalents) of Escherichia coli RNAseIII-deficient strain AB309-105expressing double-stranded RNA of target LD010 were applied to foliagesof the potato plant of variety ‘Bintje’ at the 8-12 unfolded leaf stage.Ten L1 larvae of the L. decemlineata were placed on the treated plantswith one plant per treatment. Scoring for insect mortality and growthimpediment was done on day 7 (i.e., 7 days post infestation).

As shown in FIG. 14-LD, high CPB larval mortality (90 to 100%) wasrecorded after 1 week when insects were fed potato plants treated with atopical application by fine spray of heat-inactivated cultures of E.coli harboring plasmid pGBNJO03 (for target 10 dsRNA expression) atdensities 0.25, 0.08 and 0.025 bacterial units. At 0.008 units, about athird of the insects were dead, however, the surviving insects weresignificantly smaller than those in the control groups (E. coliharbouring the empty vector pGN29 and water only). Feeding damage by CPBlarvae of the potato plant sprayed with bacteria containing the pGBNJ003plasmid at concentrations 0.025 or 0.008 units was much reduced whencompared to the damage incurred on a potato plant sprayed with bacteriacontaining the empty vector pGN29 (FIG. 15-LD).

L. Adults are Extremely Susceptible to Orally Ingested dsRNACorresponding to Target Genes

The example provided below highlights the finding that adult insects(and not only insects of the larval stage) are extremely susceptible toorally ingested dsRNA corresponding to target genes.

Four targets were chosen for this experiment: targets 2, 10, 14 and 16(SEQ ID NO: 168, 188, 198 and 220, respectively). GFP fragment dsRNA(SEQ ID NO: 235) was used as a control. Young adults (2 to 3 days old)were picked at random from our laboratory-reared culture with no biastowards insect gender. Ten adults were chosen per treatment. The adultswere prestarved for at least 6 hours before the onset of the treatment.On the first day of treatment, each adult was fed four potato leaf discs(diameter 1.5 cm²) which were pretreated with a topical application of25 μl of 0.1 μg/μl target dsRNA (synthesized as described in Example 3A;topical application as described in Example 3E) per disc. Each adult wasconfined to a small petridish (diameter 3 cm) in order to make sure thatall insects have ingested equal amounts of food and thus received equaldoses of dsRNA. The following day, each adult was again fed four treatedleaf discs as described above. On the third day, all ten adults pertreatment were collected and placed together in a cage consisting of aplastic box (dimensions 30 cm×20 cm×15 cm) with a fine nylon mesh builtinto the lid to provide good aeration. Inside the box, some moistenedfilter paper was placed in the base. Some (untreated) potato foliage wasplaced on top of the paper to maintain the adults during the experiment.From day 5, regular assessments were carried out to count the number ofdead, alive (mobile) and moribund insects. For insect moribundity,adults were laid on their backs to check whether they could rightthemselves within several minutes; an insect was considered moribundonly if it was not able to turn onto its front.

Clear specific toxic effects of double-stranded RNA corresponding todifferent targets towards adults of the Colorado potato beetle,Leptinotarsa decemlineata, were demonstrated in this experiment (FIG.12-LD). Double-stranded RNA corresponding to a gfp fragment showed notoxicity towards CPB adults on the day of the final assessment (day 19).This experiment clearly showed that the survival of CPB adults wasseverely reduced only after a few days of exposure to dsRNA whendelivered orally. For example, for target 10, on day 5, 5 out of 10adults were moribund (sick and slow moving); on day 6, 4 out of 10adults were dead with three of the survivors moribund; on day 9 alladults were observed dead.

As a consequence of this experiment, the application of targetdouble-stranded RNAs against insect pests may be broadened to includethe two life stages of an insect pest (i.e. larvae and adults) whichcould cause extensive crop damage, as is the case with the Coloradopotato beetle.

Example 4 Phaedon cochleariae (Mustard Leaf Beetle) A. Cloning of apartial sequence of the Phaedon cochleariae (mustard leaf beetle) PC001,PC003, PC005, PC010, PC014, PC016 and PC027 Genes via Family PCR

High quality, intact RNA was isolated from the third larval stage ofPhaedon cochleariae (mustard leaf beetle; source: Dr. Caroline Muller,Julius-von-Sachs-Institute for Biosciences, Chemical Ecology Group,University of Wuerzburg, Julius-von-Sachs-Platz 3, D-97082 Wuerzburg,Germany) using TRIzol Reagent (Cat. Nr. 15596-026/15596-018, Invitrogen,Rockville, Md., USA) following the manufacturer's instructions. GenomicDNA present in the RNA preparation was removed by DNase (Cat. Nr. 1700,Promega) treatment following the manufacturer's instructions. cDNA wasgenerated using a commercially available kit (SuperScript™ III ReverseTranscriptase, Cat. Nr. 18080044, Invitrogen, Rockville, Md., USA)following the manufacturer's instructions.

To isolate cDNA sequences comprising a portion of the PC001, PC003,PC005, PC010, PC014, PC016 and PC027 genes, a series of PCR reactionswith degenerate primers were performed using Amplitaq Gold (Cat. Nr.N8080240, Applied Biosystems) following the manufacturer's instructions.

The sequences of the degenerate primers used for amplification of eachof the genes are given in Table 2-PC. Table 2-PC displays Phaedoncochleariae target genes including primer sequences and cDNA sequencesobtained. These primers were used in respective PCR reactions with thefollowing conditions: 10 minutes at 95° C., followed by 40 cycles of 30seconds at 95° C., 1 minute at 55° C. and 1 minute at 72° C., followedby 10 minutes at 72° C. The resulting PCR fragments were analyzed onagarose gel, purified (QIAquick Gel Extraction kit, Cat. Nr. 28706,Qiagen), cloned into the pCR4/TOPO vector (Cat. Nr. K4530-20,Invitrogen) and sequenced. The sequences of the resulting PCR productsare represented by the respective SEQ ID NO:s as given in Table 2-PC andare referred to as the partial sequences.

The corresponding partial amino acid sequence are represented by therespective SEQ ID NO:s as given in Table 3-PC. Table 3-PC provides aminoacid sequences of cDNA clones, and the start of the reading frame isindicated in brackets.

B. dsRNA Production of the Phaedon cochleariae Genes

dsRNA was synthesized in milligram amounts using the commerciallyavailable kit T7 Ribomax™ Express RNAi System (Cat. Nr. P1700, Promega).First two separate single 5′ T7 RNA polymerase promoter templates weregenerated in two separate PCR reactions, each reaction containing thetarget sequence in a different orientation relative to the T7 promoter.

For each of the target genes, the sense T7 template was generated usingspecific T7 forward and specific reverse primers. The sequences of therespective primers for amplifying the sense template for each of thetarget genes are given in Table 8-PC. Table 8-PC provides details forpreparing ds RNA fragments of Phaedon cochleariae target sequences,including primer sequences.

The conditions in the PCR reactions were as follows: 1 minute at 95° C.,followed by 20 cycles of 30 seconds at 95° C., 30 seconds at 60° C. and1 minute at 72° C., followed by 15 cycles of 30 seconds at 95° C., 30seconds at 50° C. and 1 minute at 72° C. followed by 10 minutes at 72°C. The anti-sense T7 template was generated using specific forward andspecific T7 reverse primers in a PCR reaction with the same conditionsas described above. The sequences of the respective primers foramplifying the anti-sense template for each of the target genes aregiven in Table 8-PC. The resulting PCR products were analyzed on agarosegel and purified by PCR purification kit (Qiaquick PCR Purification Kit,Cat. Nr. 28106, Qiagen) and NaClO₄ precipitation. The generated T7forward and reverse templates were mixed to be transcribed and theresulting RNA strands were annealed, DNase and RNase treated, andpurified by sodium acetate, following the manufacturer's instructions.The sense strand of the resulting dsRNA for each of the target genes isgiven in Table 8-PC.

C. Laboratory Trials to Test dsRNA Targets, Using Oilseed Rape LeafDiscs for Activity Against Phaedon cochleariae Larvae

The example provided below is an exemplification of the finding that themustard leaf beetle (MLB) larvae are susceptible to orally ingesteddsRNA corresponding to own target genes.

To test the different double-stranded RNA samples against MLB larvae, aleaf disc assay was employed using oilseed rape (Brassica napus varietySW Oban; source: Nick Balaam, Sw Seed Ltd., 49 North Road, Abington,Cambridge, CB1 6AS, UK) leaf material as food source. The insectcultures were maintained on the same variety of oilseed rape in theinsect chamber at 25±2° C. and 60±5% relative humidity with aphotoperiod of 16 h light/8 h dark. Discs of approximately 1.1 cm indiameter (or 0.95 cm²) were cut out off leaves of 4- to 6-week old rapeplants using a suitably-sized cork borer. Double-stranded RNA sampleswere diluted to 0.1 μg/μl in Milli-Q water containing 0.05% TritonX-100. Treated leaf discs were prepared by applying 25 μl of the dilutedsolution of target PC001, PC003, PC005, PC010, PC014, PC016, PC027 dsRNAand control gfp dsRNA or 0.05% Triton X-100 on the adaxial leaf surface.The leaf discs were left to dry and placed individually in each of the24 wells of a 24-well multiplate containing 1 ml of gellified 2% agarwhich helps to prevent the leaf disc from drying out. Two neonate MLBlarvae were placed into each well of the plate, which was then coveredwith a multiwell plastic lid. The plate (one treatment containing 48insects) was divided into 4 replicates of 12 insects per replicate (eachrow). The plate containing the insects and leaf discs were kept in aninsect chamber at 25±2° C. and 60±5% relative humidity with aphotoperiod of 16 h light/8 h dark. The insects were fed leaf discs for2 days after which they were transferred to a new plate containingfreshly treated leaf discs. Thereafter, 4 days after the start of thebioassay, the insects from each replicate were collected and transferredto a Petri dish containing untreated fresh oilseed rape leaves. Larvalmortality and average weight were recorded at days 2, 4 7, 9 and 11.

P. cochleariae larvae fed on intact naked target dsRNA-treated oilseedrape leaves resulted in significant increases in larval mortalities forall targets tested, as indicated in FIG. 1( a). Tested double-strandedRNA for target PC010 led to 100% larval mortality at day 9 and fortarget PC027 at day 11. For all other targets, significantly highmortality values were reached at day 11 when compared to control gfpdsRNA, 0.05% Triton X-100 alone or untreated leaf only: (average valuein percentage±confidence interval with alpha 0.05) PC001 (94.4±8.2);PC003 (86.1±4.1); PC005 (83.3±7.8); PC014 (63.9±20.6); PC016(75.0±16.8); gfp dsRNA (11.1±8.2); 0.05% Triton X-100 (19.4±10.5); leafonly (8.3±10.5).

Larval survivors were assessed based on their average weight. For alltargets tested, the mustard leaf beetle larvae had significantly reducedaverage weights after day 4 of the bioassay; insects fed control gfpdsRNA or 0.05% Triton X-100 alone developed normally, as for the larvaeon leaf only (FIG. 1( b)-PC).

D. Laboratory Trials to Screen dsRNAs at Different Concentrations UsingOilseed Rape Leaf Discs for Activity Against Phaedon cochleariae Larvae

Twenty-five μl of a solution of dsRNA from target PC010 or PC027 atserial ten-fold concentrations from 0.1 μg/μl down to 0.1 ng/μl wasapplied topically onto the oilseed rape leaf disc, as described inExample 4D above. As a negative control, 0.05% Triton X-100 only wasadministered to the leaf disc. Per treatment, twenty-four mustard leafbeetle neonate larvae, with two insects per well, were tested. Theplates were stored in the insect rearing chamber at 25±2° C., 60±5%relative humidity, with a 16:8 hours light:dark photoperiod. At day 2,the larvae were transferred on to a new plate containing freshdsRNA-treated leaf discs. At day 4 for target PC010 and day 5 for targetPC027, insects from each replicate were transferred to a Petri dishcontaining abundant untreated leaf material. The beetles were assessedas live or dead on days 2, 4, 7, 8, 9, and 11 for target PC010, and 2,5, 8, 9 and 12 for target PC027.

Feeding oilseed rape leaf discs containing intact naked dsRNAs of thetwo different targets, PC010 and PC027, to P. cochleariae larvaeresulted in high mortalities at concentrations down to as low as 1 ngdsRNA/μl solution, as shown in FIGS. 2( a) and (b). Average mortalityvalues in percentage±confidence interval with alpha 0.05 for differentconcentrations of dsRNA for target PC010 at day 11, 0 μg/μl: 8.3±9.4;0.1 μg/μl: 100; 0.01 μg/μl: 79.2±20.6; 0.001 μg/μl: 58.3±9.4; 0.0001μg/μl: 12.5±15.6; and for target PC027 at day 12, 0 μg/μl: 8.3±9.4; 0.1μg/μl: 95.8±8.2; 0.01 μg/μl: 95.8±8.2; 0.001 μg/μl: 83.3±13.3; 0.0001μg/μl: 12.5±8.2.

E. Cloning of a MLB Gene Fragment in a Vector Suitable for BacterialProduction of Insect-Active Double-Stranded RNA

What follows is an example of cloning a DNA fragment corresponding to anMLB gene target in a vector for the expression of double-stranded RNA ina bacterial host, although any vector comprising a T7 promoter or anyother promoter for efficient transcription in bacteria, may be used(reference to WO0001846).

The sequences of the specific primers used for the amplification oftarget genes are provided in Table 8. The template used is thepCR8/GW/topo vector containing any of target sequences. The primers areused in a PCR reaction with the following conditions: 5 minutes at 98°C., followed by 30 cycles of 10 seconds at 98° C., 30 seconds at 55° C.and 2 minutes at 72° C., followed by 10 minutes at 72° C. The resultingPCR fragment is analyzed on agarose gel, purified (QIAquick GelExtraction kit, Cat. Nr. 28706, Qiagen), blunt-end cloned into SrfI-linearized pGNA49A vector (reference to WO00188121A1), and sequenced.The sequence of the resulting PCR product corresponds to the respectivesequence as given in Table 8. The recombinant vector harbouring thissequence is named pGBNJ00 (to be completed).

The sequences of the specific primers used for the amplification oftarget gene fragment PC010 are provided in Table 8-PC. The template usedwas the pCR8/GW/topo vector containing the PC010 sequence (SEQ ID NO:253). The primers were used in a touch-down PCR reaction with thefollowing conditions: 1 minute at 95° C., followed by 20 cycles of 30seconds at 95° C., 30 seconds at 60° C. with temperature decrease of−0.5° C. per cycle and 1 minute at 72° C., followed by 15 cycles of 30seconds at 95° C., 30 seconds at 50° C. and 1 minute at 72° C., followedby 10 minutes at 72° C. The resulting PCR fragment was analyzed onagarose gel, purified (QIAquick Gel Extraction kit, Cat. Nr. 28706,Qiagen), blunt-end cloned into Srf I-linearized pGNA49A vector(reference to WO00188121A1), and sequenced. The sequence of theresulting PCR product corresponds to SEQ ID NO: 488 as given in Table8-PC. The recombinant vector harbouring this sequence was namedpGCDJ001.

F. Expression and Production of a Double-Stranded RNA Target in TwoStrains of Escherichia coli AB309-105

The procedures described below are followed in order to express suitablelevels of insect-active double-stranded RNA of insect target inbacteria. In this experiment, an RNaseIII-deficient strain, AB309-105 isused.

Transformation of AB309-105

Three hundred ng of the plasmid were added to and gently mixed in a 50μl aliquot of ice-chilled chemically competent E. coli strain AB309-105.The cells were incubated on ice for 20 minutes before subjecting them toa heat shock treatment of 37° C. for 5 minutes, after which the cellswere placed back on ice for a further 5 minutes, Four hundred and fiftyμl of room temperature SOC medium was added to the cells and thesuspension incubated on a shaker (250 rpm) at 37° C. for 1 hour. Onehundred μl of the bacterial cell suspension was transferred to a 500 mlconical flask containing 150 ml of liquid Luria-Bertani (LB) brothsupplemented with 100 μg/ml carbenicillin antibiotic. The culture wasincubated on an Innova 4430 shaker (250 rpm) at 37° C. overnight (16 to18 hours).

Chemical Induction of Double-Stranded RNA Expression in AB309-105

Expression of double-stranded RNA from the recombinant vector,pGBNJ0003, in the bacterial strain AB309-105 was made possible since allthe genetic components for controlled expression are present. In thepresence of the chemical inducer isopropylthiogalactoside, or IPTG, theT7 polymerase will drive the transcription of the target sequence inboth antisense and sense directions since these are flanked byoppositely oriented T7 promoters.

The optical density at 600 nm of the overnight bacterial culture wasmeasured using an appropriate spectrophotometer and adjusted to a valueof 1 by the addition of fresh LB broth. Fifty ml of this culture wastransferred to a 50 ml Falcon tube and the culture then centrifuged at3000 g at 15° C. for 10 minutes. The supernatant was removed and thebacterial pellet resuspended in 50 ml of fresh S complete medium (SNCmedium plus 5 μg/ml cholesterol) supplemented with 100 μg/mlcarbenicillin and 1 mM IPTG. The bacteria were induced for 2 to 4 hoursat room temperature.

Heat Treatment of Bacteria

Bacteria were killed by heat treatment in order to minimize the risk ofcontamination of the artificial diet in the test plates. However, heattreatment of bacteria expressing double-stranded RNA is not aprerequisite for inducing toxicity towards the insects due to RNAinterference. The induced bacterial culture was centrifuged at 3000 g atroom temperature for 10 minutes, the supernatant discarded and thepellet subjected to 80° C. for 20 minutes in a water bath. After heattreatment, the bacterial pellet was resuspended in a total volume of 50ml of 0.05% Triton X-100 solution. The tube was stored at 4° C. untilfurther use

G. Laboratory Trials to Test Escherichia coli Expressing dsRNA TargetsAgainst Phaedon cochleariae Leaf Disc Bioassays

The leaf-disc bioassay method was employed to test double-stranded RNAfrom target PC010 produced in Escherichia coli (from plasmid pGCDJ001)against larvae of the mustard leaf beetle. Leaf discs were prepared fromoilseed rape foliage, as described in Example 4. Twenty μl of abacterial suspension, with an optical density measurement of 1 at 600 nmwavelength, was pipetted onto each disc. The leaf disc was placed in awell of a 24-multiwell plate containing 1 ml gellified agar. On eachleaf disc were added two neonate larvae. For each treatment, 3replicates of 16 neonate larvae per replicate were prepared. The plateswere kept in the insect rearing chamber at 25±2° C. and 60±5% relativehumidity, with a 16:8 hours light:dark photoperiod. At day 3 (i.e. 3days post start of bioassay), larvae were transferred to a new platecontaining fresh treated (same dosage) leaf discs. The leaf material wasrefreshed every other day from day 5 onwards. The bioassay was scored onmortality and average weight. Negative controls were leaf discs treatedwith bacteria harbouring plasmid pGN29 (empty vector) and leaf only.

A clear increase in mortality of P. cochleariae larvae with time wasshown after the insects were fed on oilseed rape leaves treated with asuspension of RNaseIII-deficient E. coli strain AB309-105 containingplasmid pGCDJ001, whereas very little or no insect mortality wasobserved in the case of bacteria with plasmid pGN29 or leaf only control(FIG. 3-PC).

Plant-Based Bioassays

Whole plants are sprayed with suspensions of chemically induced bacteriaexpressing dsRNA prior to feeding the plants to MLB. The are grown fromin a plant growth room chamber. The plants are caged by placing a 500 mlplastic bottle upside down over the plant with the neck of the bottlefirmly placed in the soil in a pot and the base cut open and coveredwith a fine nylon mesh to permit aeration, reduce condensation insideand prevent insect escape. MLB are placed on each treated plant in thecage. Plants are treated with a suspension of E. coli AB309-105harbouring the pGBNJ001 plasmids or pGN29 plasmid. Different quantitiesof bacteria are applied to the plants: for instance 66, 22, and 7 units,where one unit is defined as 10⁹ bacterial cells in 1 ml of a bacterialsuspension at optical density value of 1 at 600 nm wavelength. In eachcase, a total volume of between 1 and 10 ml s sprayed on the plant withthe aid of a vaporizer. One plant is used per treatment in this trial.The number of survivors are counted and the weight of each survivorrecorded.

Spraying plants with a suspension of E. coli bacterial strain AB309-105expressing target dsRNA from pGBNJ003 lead to a dramatic increase ininsect mortality when compared to pGN29 control. These experiments showthat double-stranded RNA corresponding to an insect gene target sequenceproduced in either wild-type or RNaseIII-deficient bacterial expressionsystems is toxic towards the insect in terms of substantial increases ininsect mortality and growth/development delay for larval survivors. Itis also clear from these experiments that an exemplification is providedfor the effective protection of plants/crops from insect damage by theuse of a spray of a formulation consisting of bacteria expressingdouble-stranded RNA corresponding to an insect gene target.

Example 5 Epilachna varivetis (Mexican Bean Beetle) A. Cloning Epilachnavarivetis Partial Gene Sequences

High quality, intact RNA was isolated from 4 different larval stages ofEpilachna varivetis (Mexican bean beetle; source: Thomas Dorsey,Supervising Entomologist, New Jersey Department of Agriculture, Divisionof Plant Industry, Bureau of Biological Pest Control, Phillip AlampiBeneficial Insect Laboratory, PO Box 330, Trenton, N.J. 08625-0330, USA)using TRIzol Reagent (Cat. Nr. 15596-026/15596-018, Invitrogen,Rockville, Md., USA) following the manufacturer's instructions. GenomicDNA present in the RNA preparation was removed by DNase treatmentfollowing the manufacturer's instructions (Cat. Nr. 1700, Promega). cDNAwas generated using a commercially available kit (SuperScript™ IIIReverse Transcriptase, Cat. Nr. 18080044, Invitrogen, Rockville, Md.,USA) following the manufacturer's instructions.

To isolate cDNA sequences comprising a portion of the EV005, EV009,EV010, EV015 and EV016 genes, a series of PCR reactions with degenerateprimers were performed using Amplitaq Gold (Cat. Nr. N8080240, AppliedBiosystems) following the manufacturer's instructions.

The sequences of the degenerate primers used for amplification of eachof the genes are given in Table 2-EV, which displays Epilachna varivetistarget genes including primer sequences and cDNA sequences obtained.These primers were used in respective PCR reactions with the followingconditions: for EV005 and EV009, 10 minutes at 95° C., followed by 40cycles of 30 seconds at 95° C., 1 minute at 50° C. and 1 minute 30seconds at 72° C., followed by 7 minutes at 72° C.; for EV014, 10minutes at 95° C., followed by 40 cycles of 30 seconds at 95° C., 1minute at 53° C. and 1 minute at 72° C., followed by 7 minutes at 72°C.; for EV010 and EV016, 10 minutes at 95° C., followed by 40 cycles of30 seconds at 95° C., 1 minute at 54° C. and 1 minute 40 seconds at 72°C., followed by 7 minutes at 72° C. The resulting PCR fragments wereanalyzed on agarose gel, purified (QIAquick Gel Extraction kit, Cat. Nr.28706, Qiagen), cloned into the pCR4/TOPO vector (Cat. Nr. K4530-20,Invitrogen), and sequenced. The sequences of the resulting PCR productsare represented by the respective SEQ ID NO:s as given in Table 2-EV andare referred to as the partial sequences. The corresponding partialamino acid sequences are represented by the respective SEQ ID NO:s asgiven in Table 3-EV, where the start of the reading frame is indicatedin brackets.

B. dsRNA Production of the Epilachna varivetis Genes

dsRNA was synthesized in milligram amounts using the commerciallyavailable kit T7 Ribomax™ Express RNAi System (Cat. Nr. P1700, Promega).First two separate single 5′ T7 RNA polymerase promoter templates weregenerated in two separate PCR reactions, each reaction containing thetarget sequence in a different orientation relative to the T7 promoter.

For each of the target genes, the sense T7 template was generated usingspecific T7 forward and specific reverse primers. The sequences of therespective primers for amplifying the sense template for each of thetarget genes are given in Table 8-EV.

The conditions in the PCR reactions were as follows: 1 minute at 95° C.,followed by 20 cycles of 30 seconds at 95° C., 30 seconds at 60° C. and1 minute at 72° C., followed by 15 cycles of 30 seconds at 95° C., 30seconds at 50° C. and 1 minute at 72° C. followed by 10 minutes at 72°C. The anti-sense T7 template was generated using specific forward andspecific T7 reverse primers in a PCR reaction with the same conditionsas described above. The sequences of the respective primers foramplifying the anti-sense template for each of the target genes aregiven in Table 8-EV. The resulting PCR products were analyzed on agarosegel and purified by PCR purification kit (Qiaquick PCR Purification Kit,Cat., Nr. 28106, Qiagen) and NaClO₄ precipitation. The generated T7forward and reverse templates were mixed to be transcribed and theresulting RNA strands were annealed, DNase and RNase treated, andpurified by sodium acetate, following the manufacturer's instructions.The sense strand of the resulting dsRNA for each of the target genes isgiven in Table 8-EV.

C. Laboratory Trials to Test dsRNA Targets Using Bean Leaf Discs forActivity Against Epilachna varivetis Larvae

The example provided below is an exemplification of the finding that theMexican bean beetle (MBB) larvae are susceptible to orally ingesteddsRNA corresponding to own target genes.

To test the different double-stranded RNA samples against MBB larvae, aleaf disc assay was employed using snap bean (Phaseolus vulgaris varietyMontano; source: Aveve NV, Belgium) leaf material as food source. Thesame variety of beans was used to maintain insect cultures in the insectchamber at 25±2° C. and 60±5% relative humidity with a photoperiod of 16h light/8 h dark. Discs of approximately 1.1 cm in diameter (or 0.95cm²) were cut out off leaves of 1- to 2-week old bean plants using asuitably-sized cork borer. Double-stranded RNA samples were diluted to 1μg/μl in Milli-Q water containing 0.05% Triton X-100. Treated leaf discswere prepared by applying 25 μl of the diluted solution of target Ev005,Ev010, Ev015, Ev016 dsRNA and control gfp dsRNA or 0.05% Triton X-100 onthe adaxial leaf surface. The leaf discs were left to dry and placedindividually in each of the 24 wells of a 24-well multiplate containing1 ml of gellified 2% agar which helps to prevent the leaf disc fromdrying out. A single neonate MBB larva was placed into each well of aplate, which was then covered with a multiwell plastic lid. The platewas divided into 3 replicates of 8 insects per replicate (row). Theplate containing the insects and leaf discs were kept in an insectchamber at 25±2° C. and 60±5% relative humidity with a photoperiod of 16h light/8 h dark. The insects were fed on the leaf discs for 2 daysafter which the insects were transferred to a new plate containingfreshly treated leaf discs. Thereafter, 4 days after the start of thebioassay, the insects were transferred to a petriplate containinguntreated fresh bean leaves every day until day 10. Insect mortality wasrecorded at day 2 and every other day thereafter.

Feeding snap bean leaves containing surface-applied intact naked targetdsRNAs to E. varivestis larvae resulted in significant increases inlarval mortalities, as indicated in FIG. 1. Tested double-stranded RNAsof targets Ev010, Ev015, & Ev016 led to 100% mortality after 8 days,whereas dsRNA of target Ev005 took 10 days to kill all larvae. Themajority of the insects fed on treated leaf discs containing control gfpdsRNA or only the surfactant Triton X-100 were sustained throughout thebioassay (FIG. 1-EV).

D. Laboratory Trials to Test dsRNA Targets Using Bean Leaf Discs forActivity Against Epilachna varivestis Adults

The example provided below is an exemplification of the finding that theMexican bean beetle adults are susceptible to orally ingested dsRNAcorresponding to own target genes.

In a similar bioassay set-up as for Mexican bean beetle larvae, adultMBBs were tested against double-stranded RNAs topically-applied to beanleaf discs. Test dsRNA from each target Ev010, Ev015 and Ev016 wasdiluted in 0.05% Triton X-100 to a final concentration of 0.1 μg/μl.Bean leaf discs were treated by topical application of 30 μl of the testsolution onto each disc. The discs were allowed to dry completely beforeplacing each on a slice of gellified 2% agar in each well of a 24-wellmultiwell plate. Three-day-old adults were collected from the culturecages and fed nothing for 7-8 hours prior to placing one adult to eachwell of the bioassay plate (thus 24 adults per treatment). The plateswere kept in the insect rearing chamber (under the same conditions asfor MBB larvae for 24 hours) after which the adults were transferred toa new plate containing fresh dsRNA-treated leaf discs. After a further24 hours, the adults from each treatment were collected and placed in aplastic box with dimensions 30 cm×15 cm×10 cm containing two potted anduntreated 3-week-old bean plants. Insect mortality was assessed from day4 until day 11.

All three target dsRNAs (Ev010, Ev015 and Ev016) ingested by adults ofEpilachna varivestis resulted in significant increases in mortality fromday 4 (4 days post bioassay start), as shown in FIG. 2( a)-EV. From day5, dramatic changes in feeding patterns were observed between insectsfed initially with target-dsRNA-treated bean leaf discs and those thatwere fed discs containing control gfp dsRNA or surfactant Triton X-100.Reductions in foliar damage by MBB adults of untreated bean plants wereclearly visible for all three targets when compared to gfp dsRNA andsurfactant only controls, albeit at varying levels; insects fed target15 caused the least damage to bean foliage (FIG. 2( b)-EV).

E. Cloning of a MBB Gene Fragment in a Vector Suitable for BacterialProduction of Insect-Active Double-Stranded RNA

What follows is an example of cloning a DNA fragment corresponding to anMLB gene target in a vector for the expression of double-stranded RNA ina bacterial host, although any vector comprising a T7 promoter or anyother promoter for efficient transcription in bacteria, may be used(reference to WO0001846).

The sequences of the specific primers used for the amplification oftarget genes are provided in Table 8-EV. The template used is thepCR8/GW/topo vector containing any of target sequences. The primers areused in a PCR reaction with the following conditions: 5 minutes at 98°C., followed by 30 cycles of 10 seconds at 98° C., 30 seconds at 55° C.and 2 minutes at 72° C., followed by 10 minutes at 72° C. The resultingPCR fragment is analyzed on agarose gel, purified (QIAquick GelExtraction kit, Cat. Nr. 28706, Qiagen), blunt-end cloned into SrfI-linearized pGNA49A vector (reference to WO00188121A1), and sequenced.The sequence of the resulting PCR product corresponds to the respectivesequence as given in Table 8-EV. The recombinant vector harbouring thissequence is named pGBNJ00XX.

F. Expression and Production of a Double-Stranded RNA Target in TwoStrains of Escherichia coli: (1) AB309-105, and, (2) BL21(DE3)

The procedures described below are followed in order to express suitablelevels of insect-active double-stranded RNA of insect target inbacteria. An RNaseIII-deficient strain, AB309-105, is used in comparisonto wild-type RNaseIII-containing bacteria, BL21(DE3).

Transformation of AB309-105 and BL21 (DE3)

Three hundred ng of the plasmid are added to and gently mixed in a 50 μlaliquot of ice-chilled chemically competent E. coli strain AB309-105 orBL21(DE3). The cells are incubated on ice for 20 minutes beforesubjecting them to a heat shock treatment of 37° C. for 5 minutes, afterwhich the cells are placed back on ice for a further 5 minutes. Fourhundred and fifty μl of room temperature SOC medium is added to thecells and the suspension incubated on a shaker (250 rpm) at 37° C. for 1hour. One hundred μl of the bacterial cell suspension is transferred toa 500 ml conical flask containing 150 ml of liquid Luria-Bertani (LB)broth supplemented with 100 μg/ml carbenicillin antibiotic. The cultureis incubated on an Innova 4430 shaker (250 rpm) at 37° C. overnight (16to 18 hours).

Chemical Induction of Double-Stranded RNA Expression in AB309-105 andBL21 (DE3)

Expression of double-stranded RNA from the recombinant vector, pGBNJ003,in the bacterial strain AB309-105 or BL21(DE3) is made possible sinceall the genetic components for controlled expression are present. In thepresence of the chemical inducer isopropylthiogalactoside, or IPTG, theT 7 polymerase will drive the transcription of the target sequence inboth antisense and sense directions since these are flanked byoppositely oriented T7 promoters.

The optical density at 600 nm of the overnight bacterial culture ismeasured using an appropriate spectrophotometer and adjusted to a valueof 1 by the addition of fresh LB broth. Fifty ml of this culture istransferred to a 50 ml Falcon tube and the culture then centrifuged at3000 g at 15° C. for 10 minutes. The supernatant is removed and thebacterial pellet resuspended in 50 ml of fresh S complete medium (SNCmedium plus 5 μg/ml cholesterol) supplemented with 100 μg/mlcarbenicillin and 1 mM IPTG. The bacteria are induced for 2 to 4 hoursat room temperature.

Heat Treatment of Bacteria

Bacteria are killed by heat treatment in order to minimize the risk ofcontamination of the artificial diet in the test plates. However, heattreatment of bacteria expressing double-stranded RNA is not aprerequisite for inducing toxicity towards the insects due to RNAinterference. The induced bacterial culture is centrifuged at 3000 g atroom temperature for 10 minutes, the supernatant discarded and thepellet subjected to 80° C. for 20 minutes in a water bath. After heattreatment, the bacterial pellet is resuspended in 1.5 ml MilliQ waterand the suspension transferred to a microfuge tube. Several tubes areprepared and used in the bioassays for each refreshment. The tubes arestored at −20° C. until further use.

G. Laboratory Trials to Test Escherichia coli Expressing dsRNA TargetsAgainst Epilachna varivetis Plant-Based Bioassays

Whole plants are sprayed with suspensions of chemically induced bacteriaexpressing dsRNA prior to feeding the plants to MBB. The are grown fromin a plant growth room chamber. The plants are caged by placing a 500 mlplastic bottle upside down over the plant with the neck of the bottlefirmly placed in the soil in a pot and the base cut open and coveredwith a fine nylon mesh to permit aeration, reduce condensation insideand prevent insect escape. MMB are placed on each treated plant in thecage. Plants are treated with a suspension of E. coli AB309-105harbouring the pGBNJ001 plasmids or pGN29 plasmid. Different quantitiesof bacteria are applied to the plants: for instance 66, 22, and 7 units,where one unit is defined as 10⁹ bacterial cells in 1 ml of a bacterialsuspension at optical density value of 1 at 600 nm wavelength. In eachcase, a total volume of between 1 and 10 ml sprayed on the plant withthe aid of a vaporizer. One plant is used per treatment in this trial.The number of survivors are counted and the weight of each survivorrecorded.

Spraying plants with a suspension of E. coli bacterial strain AB309-105expressing target dsRNA from pGBNJO03 lead to a dramatic increase ininsect mortality when compared to pGN29 control. These experiments showthat double-stranded RNA corresponding to an insect gene target sequenceproduced in either wild-type or RNaseIII-deficient bacterial expressionsystems is toxic towards the insect in terms of substantial increases ininsect mortality and growth/development delay for larval survivors. Itis also clear from these experiments that an exemplification is providedfor the effective protection of plants/crops from insect damage by theuse of a spray of a formulation consisting of bacteria expressingdouble-stranded RNA corresponding to an insect gene target.

Example 6 Anthonomus grandis (Cotton Boll Weevil) A. Cloning Anthonomusgrandis Partial Sequences

High quality, intact RNA was isolated from the 3 instars of Anthonomusgrandis (cotton boll weevil; source: Dr. Gary Benzon, Benzon ResearchInc., 7 Kuhn Drive, Carlisle, Pa. 17013, USA) using TRIzol Reagent (Cat.Nr. 15596-026/15596-018, Invitrogen, Rockville, Md., USA) following themanufacturer's instructions. Genomic DNA present in the RNA preparationwas removed by DNase treatment following the manufacturer's instructions(Cat. Nr. 1700, Promega). cDNA was generated using a commerciallyavailable kit (SuperScript™ III Reverse Transcriptase, Cat. Nr.18080044, Invitrogen, Rockville, Md., USA) following the manufacturer'sinstructions.

To isolate cDNA sequences comprising a portion of the AG001, AG005,AG010, AG014 and AG016 genes, a series of PCR reactions with degenerateprimers were performed using Amplitaq Gold (Cat. Nr. N8080240, AppliedBiosystems) following the manufacturer's instructions.

The sequences of the degenerate primers used for amplification of eachof the genes are given in Table 2-AG. These primers were used inrespective PCR reactions with the following conditions: for AG001, AG005and AG016, 10 minutes at 95° C., followed by 40 cycles of 30 seconds at95° C., 1 minute at 50° C. and 1 minute and 30 seconds at 72° C.,followed by 7 minutes at 72° C.; for AG100, 10 minutes at 95° C.,followed by 40 cycles of 30 seconds at 95° C., 1 minute at 54° C. and 2minutes and 30 seconds at 72° C., followed by 7 minutes at 72° C.; forAG014, 10 minutes at 95° C., followed by 40 cycles of 30 seconds at 95°C., 1 minute at 55° C. and 1 minute at 72° C., followed by 7 minutes at72° C. The resulting PCR fragments were analyzed on agarose gel,purified (QIAquick Gel Extraction kit, Cat. Nr. 28706, Qiagen), clonedinto the pCR8/GW/TOPO vector (Cat. Nr. K2500-20, Invitrogen) andsequenced. The sequences of the resulting PCR products are representedby the respective SEQ ID NO:s as given in Table 2-AG and are referred toas the partial sequences. The corresponding partial amino acid sequenceare represented by the respective SEQ ID NO:s as given in Table 3-AG.

B. dsRNA Production of the Anthonomus grandis (Cotton Boll Weevil) Genes

dsRNA was synthesized in milligram amounts using the commerciallyavailable kit T7 Ribomax™ Express RNAi System (Cat. Nr. P1700, Promega).First two separate single 5′ T7 RNA polymerase promoter templates weregenerated in two separate PCR reactions, each reaction containing thetarget sequence in a different orientation relative to the T7 promoter.For each of the target genes, the sense T7 template was generated usingspecific T7 forward and specific reverse primers. The sequences of therespective primers for amplifying the sense template for each of thetarget genes are given in Table 8-AG. A touchdown PCR was performed asfollows: 1 minute at 95° C., followed by 20 cycles of 30 seconds at 95°C., 30 seconds at 60° C. with a decrease in temperature of 0.5° C. percycle and 1 minute at 72° C., followed by 15 cycles of 30 seconds at 95°C., 30 seconds at 50° C. and 1 minute at 72° C., followed by 10 minutesat 72° C. The anti-sense T7 template was generated using specificforward and specific T7 reverse primers in a PCR reaction with the sameconditions as described above. The sequences of the respective primersfor amplifying the anti-sense template for each of the target genes aregiven in Table 8-AG. The resulting PCR products were analyzed on agarosegel and purified by PCR purification kit (Qiaquick PCR Purification Kit,Cat. Nr. 28106, Qiagen) and NaClO₄ precipitation. The generated T7forward and reverse templates were mixed to be transcribed and theresulting RNA strands were annealed, DNase and RNase treated, andpurified by sodium acetate, following the manufacturer's instructions.The sense strand of the resulting dsRNA for each of the target genes isgiven in Table 8-AG.

C. Laboratory Trials to Test dsRNA Targets, Using Artificial Diet forActivity Against the Larvae of the House Cricket, Acheta domesticus

House crickets, Acheta domesticus, were maintained at InsectInvestigations Ltd. (origin: Blades Biological Ltd., Kent, UK). Theinsects were reared on bran pellets and cabbage leaves. Mixed sex nymphsof equal size and no more than 5 days old were selected for use in thetrial. Double-stranded RNA was mixed with a wheat-based pelleted rodentdiet (rat and mouse standard diet, B & K Universal Ltd., Grimston,Aldbrough, Hull, UK). The diet, BK001P, contains the followingingredients in descending order by weight: wheat, soya, wheatfeed,barley, pellet binder, rodent 5 vit min, fat blend, dicalcium phosphate,mould carb. The pelleted rodent diet was finely ground and heat-treatedin a microwave oven prior to mixing, in order to inactivate any enzymecomponents. All rodent diet was taken from the same batch in order toensure consistency. The ground diet and dsRNA were mixed thoroughly andformed into small pellets of equal weight, which were allowed to dryovernight at room temperature.

Double-stranded RNA samples from targets and gfp control atconcentrations 10 μg/μl are applied in the ratio 1 g ground diet plus 1ml dsRNA solution, thereby resulting in an application rate of 10 mgdsRNA per g pellet. Pellets are replaced weekly. The insects areprovided with treated pellets for the first three weeks of the trial.Thereafter untreated pellets are provided. Insects are maintained withinlidded plastic containers (9 cm diameter, 4.5 cm deep), ten percontainer. Each arena contains one treated bait pellet and one watersource (damp cotton wool ball), each placed in a separate small weighboat. The water is replenished ad lib throughout the experiment.

Assessments are made at twice weekly intervals, with no more than fourdays between assessments, until all the control insects had either diedor moulted to the adult stage (84 days). At each assessment the insectsare assessed as live or dead, and examined for abnormalities. From day46 onwards, once moulting to adult commences, all insects (live anddead) are assessed as nymph or adult. Surviving insects are weighed onday 55 of the trial. Four replicates are performed for each of thetreatments. During the trial the test conditions are 25 to 33° C. and 20to 25% relative humidity, with a 12:12 hour light:dark photoperiod.

D. Cloning of a MLB Gene Fragment in a Vector Suitable for BacterialProduction of Insect-Active Double-Stranded RNA

What follows is an example of cloning a DNA fragment corresponding to anMLB gene target in a vector for the expression of double-stranded RNA ina bacterial host, although any vector comprising a T7 promoter or anyother promoter for efficient transcription in bacteria, may be used(reference to WO0001846).

The sequences of the specific primers used for the amplification oftarget genes are provided in Table 8. The template used is thepCR8/GW/topo vector containing any of target sequences. The primers areused in a PCR reaction with the following conditions: 5 minutes at 98°C., followed by 30 cycles of 10 seconds at 98° C., 30 seconds at 55° C.and 2 minutes at 72° C., followed by 10 minutes at 72° C. The resultingPCR fragment is analyzed on agarose gel, purified (QIAquick GelExtraction kit, Cat. Nr. 28706, Qiagen), blunt-end cloned into SrfI-linearized pGNA49A vector (reference to WO00188121A1), and sequenced.The sequence of the resulting PCR product corresponds to the respectivesequence as given in Table 8. The recombinant vector harbouring thissequence is named pGBNJ00XX.

E. Expression and production of a double-stranded RNA target in twostrains of Escherichia coli: (1) AB309-105, and, (2) BL21(DE3)

The procedures described below are followed in order to express suitablelevels of insect-active double-stranded RNA of insect target inbacteria. An RNaseIII-deficient strain, AB309-105, is used in comparisonto wild-type RNaseIII-containing bacteria, BL21(DE3).

Transformation of AB309-105 and BL21(DE3)

Three hundred ng of the plasmid are added to and gently mixed in a 50 μlaliquot of ice-chilled chemically competent E. coli strain AB309-105 orBL21(DE3). The cells are incubated on ice for 20 minutes beforesubjecting them to a heat shock treatment of 37° C. for 5 minutes, afterwhich the cells are placed back on ice for a further 5 minutes. Fourhundred and fifty μl of room temperature SOC medium is added to thecells and the suspension incubated on a shaker (250 rpm) at 37° C. for 1hour. One hundred μl of the bacterial cell suspension is transferred toa 500 ml conical flask containing 150 ml of liquid Luria-Bertani (LB)broth supplemented with 100 μg/ml carbenicillin antibiotic. The cultureis incubated on an Innova 4430 shaker (250 rpm) at 37° C. overnight (16to 18 hours).

Chemical Induction of Double-Stranded RNA Expression in AB309-105 andBL21(DE3)

Expression of double-stranded RNA from the recombinant vector, pGBNJ003,in the bacterial strain AB309-105 or BL21(DE3) is made possible sinceall the genetic components for controlled expression are present. In thepresence of the chemical inducer isopropylthiogalactoside, or IPTG, theT7 polymerase will drive the transcription of the target sequence inboth antisense and sense directions since these are flanked byoppositely oriented T7 promoters.

The optical density at 600 nm of the overnight bacterial culture ismeasured using an appropriate spectrophotometer and adjusted to a valueof 1 by the addition of fresh LB broth. Fifty ml of this culture istransferred to a 50 ml Falcon tube and the culture then centrifuged at3000 g at 15° C. for 10 minutes, The supernatant is removed and thebacterial pellet resuspended in 50 ml of fresh S complete medium (SNCmedium plus 5 μg/ml cholesterol) supplemented with 100 μg/mlcarbenicillin and 1 mM IPTG. The bacteria are induced for 2 to 4 hoursat room temperature.

Heat Treatment of Bacteria

Bacteria are killed by heat treatment in order to minimise the risk ofcontamination of the artificial diet in the test plates. However, heattreatment of bacteria expressing double-stranded RNA is not aprerequisite for inducing toxicity towards the insects due to RNAinterference. The induced bacterial culture is centrifuged at 3000 g atroom temperature for 10 minutes, the supernatant discarded and thepellet subjected to 80° C. for 20 minutes in a water bath. After heattreatment, the bacterial pellet is resuspended in 1.5 ml MilliQ waterand the suspension transferred to a microfuge tube. Several tubes areprepared and used in the bioassays for each refreshment. The tubes arestored at −20° C. until further use.

F. Laboratory Trials to Test Escherichia coli Expressing dsRNA TargetsAgainst Anthonomus grandis Plant-Based Bioassays

Whole plants are sprayed with suspensions of chemically induced bacteriaexpressing dsRNA prior to feeding the plants to CBW. The are grown fromin a plant growth room chamber. The plants are caged by placing a 500 mlplastic bottle upside down over the plant with the neck of the bottlefirmly placed in the soil in a pot and the base cut open and coveredwith a fine nylon mesh to permit aeration, reduce condensation insideand prevent insect escape. CBW are placed on each treated plant in thecage. Plants are treated with a suspension of E. coli AB309-105harbouring the pGBNJO01 plasmids or pGN29 plasmid. Different quantitiesof bacteria are applied to the plants: for instance 66, 22, and 7 units,where one unit is defined as 10⁹ bacterial cells in 1 ml of a bacterialsuspension at optical density value of 1 at 600 nm wavelength. In eachcase, a total volume of between 1 and 10 ml s sprayed on the plant withthe aid of a vaporizer. One plant is used per treatment in this trial.The number of survivors are counted and the weight of each survivorrecorded.

Spraying plants with a suspension of E. coli bacterial strain AB309-105expressing target dsRNA from pGBNJO03 lead to a dramatic increase ininsect mortality when compared to pGN29 control. These experiments showthat double-stranded RNA corresponding to an insect gene target sequenceproduced in either wild-type or RNaseIII-deficient bacterial expressionsystems is toxic towards the insect in terms of substantial increases ininsect mortality and growth/development delay for larval survivors. Itis also clear from these experiments that an exemplification is providedfor the effective protection of plants/crops from insect damage by theuse of a spray of a formulation consisting of bacteria expressingdouble-stranded RNA corresponding to an insect gene target.

Example 7 Tribolium castaneum (Red Flour Beetle) A. Cloning Triboliumcastaneum Partial Sequences

High quality, intact RNA was isolated from all the different insectstages of Tribolium castaneum (red flour beetle; source: Dr. LaraSenior, Insect Investigations Ltd., Capital Business Park, Wentloog,Cardiff, CF3 2PX, Wales, UK) using TRIzol Reagent (Cat. Nr.15596-026/15596-018, Invitrogen, Rockville, Md., USA) following themanufacturer's instructions. Genomic DNA present in the RNA preparationwas removed by DNase treatment following the manufacturer's instructions(Cat. Nr. 1700, Promega). cDNA was generated using a commerciallyavailable kit (SuperScript™ III Reverse Transcriptase, Cat. Nr.18080044, Invitrogen, Rockville, Md., USA) following the manufacturer'sinstructions.

To isolate cDNA sequences comprising a portion of the TC001, TC002,TC010, TC014 and TC015 genes, a series of PCR reactions with degenerateprimers were performed using Amplitaq Gold (Cat. Nr. N8080240, AppliedBiosystems) following the manufacturer's instructions.

The sequences of the degenerate primers used for amplification of eachof the genes are given in Table 2-TC. These primers were used inrespective PCR reactions with the following conditions: 10 minutes at95° C., followed by 40 cycles of 30 seconds at 95° C., 1 minute at 50°C. and 1 minute and 30 seconds at 72° C., followed by 7 minutes at 72°C. (TC001, TC014, TC015); 10 minutes at 95° C., followed by 40 cycles of30 seconds at 95° C., 1 minute at 54° C. and 2 minutes and 30 seconds at72° C., followed by 7 minutes at 72° C. (TC010); 10 minutes at 95° C.,followed by 40 cycles of 30 seconds at 95° C., 1 minute at 53° C. and 1minute at 72° C., followed by 7 minutes at 72° C. (TC002). The resultingPCR fragments were analyzed on agarose gel, purified (QIAquick GelExtraction kit, Cat. Nr. 28706, Qiagen), cloned into the pCR8/GW/TOPOvector (Cat. Nr. K2500-20, Invitrogen), and sequenced. The sequences ofthe resulting PCR products are represented by the respective SEQ ID NO:sas given in Table 2-TC and are referred to as the partial sequences. Thecorresponding partial amino acid sequences are represented by therespective SEQ ID NO:s as given in Table 3-TC.

B. dsRNA Production of the Tribolium castaneum Genes

dsRNA was synthesized in milligram amounts using the commerciallyavailable kit T7 Ribomax™ Express RNAi System (Cat. Nr. P1700, Promega).First two separate single 5′ T7 RNA polymerase promoter templates weregenerated in two separate PCR reactions, each reaction containing thetarget sequence in a different orientation relative to the T7 promoter.

For each of the target genes, the sense T7 template was generated usingspecific T7 forward and specific reverse primers. The sequences of therespective primers for amplifying the sense template for each of thetarget genes are given in Table 8-TC. The conditions in the PCRreactions were as follows: 1 minute at 95° C., followed by 20 cycles of30 seconds at 95° C., 30 seconds at 60° C. (−0.5° C./cycle) and 1 minuteat 72° C., followed by 15 cycles of 30 seconds at 95° C., 30 seconds at50° C. and 1 minute at 72° C., followed by 10 minutes at 72° C. Theanti-sense T7 template was generated using specific forward and specificT7 reverse primers in a PCR reaction with the same conditions asdescribed above. The sequences of the respective primers for amplifyingthe anti-sense template for each of the target genes are given in Table8-TC. The resulting PCR products were analyzed on agarose gel andpurified by PCR purification kit (Qiaquick PCR Purification Kit, Cat.Nr. 28106, Qiagen) and NaClO₄ precipitation. The generated T7 forwardand reverse templates were mixed to be transcribed and the resulting RNAstrands were annealed, DNase and RNase treated, and purified by sodiumacetate, following the manufacturer's instructions. The sense strand ofthe resulting dsRNA for each of the target genes is given in Table 8-TC.

C. Laboratory Trials to Test dsRNA Targets, Using Artificial Diet forActivity Against Tribolium castaneum Larvae

The example provided below is an exemplification of the finding that thered flour beetle (RFB) larvae are susceptible to orally ingested dsRNAcorresponding to own target genes.

Red flour beetles, Tribolium castaneum, were maintained at InsectInvestigations Ltd. (origin: Imperial College of Science, Technology andMedicine, Silwood Park, Berkshire, UK). Insects were cultured accordingto company SOP/251/01. Briefly, the beetles were housed in plastic jarsor tanks. These have an open top to allow ventilation. A piece ofnetting was fitted over the top and secured with an elastic band toprevent escape. The larval rearing medium (flour) was placed in thecontainer where the beetles can breed. The stored product beetlecolonies were maintained in a controlled temperature room at 25±3° C.with a 16:8 hour light:dark cycle.

Double-stranded RNA from target TC014 (with sequence corresponding toSEQ D NO: -799) was incorporated into a mixture of flour and milk powder(wholemeal flour: powdered milk in the ratio 4:1) and left to dryovernight. Each replicate was prepared separately: 100 μl of a 10 μg/μldsRNA solution (1 mg dsRNA) was added to 0.1 g flour/milk mixture. Thedried mixture was ground to a fine powder. Insects were maintainedwithin Petri dishes (55 mm diameter), lined with a double layer offilter paper. The treated diet was placed between the two filter paperlayers. Ten first instar, mixed sex larvae were placed in each dish(replicate). Four replicates were performed for each treatment. Controlwas Milli-Q water. Assessments (number of survivors) were made on aregular basis. During the trial, the test conditions were 25-33° C. and20-25% relative humidity, with a 12:12 hour light:dark photoperiod.

Survival of larvae of T. castaneum over time on artificial diet treatedwith target TC014 dsRNA was significantly reduced when compared to dietonly control, as shown in FIG. 1.

D. Cloning of a RFB Gene Fragment in a Vector Suitable for BacterialProduction of Insect-Active Double-Stranded RNA

What follows is an example of cloning a DNA fragment corresponding to anRFB gene target in a vector for the expression of double-stranded RNA ina bacterial host, although any vector comprising a T7 promoter or anyother promoter for efficient transcription in bacteria, may be used(reference to WO0001846).

The sequences of the specific primers used for the amplification oftarget genes are provided in Table 8-TC. The template used is thepCR8/GW/topo vector containing any of target sequences. The primers areused in a PCR reaction with the following conditions: 5 minutes at 98°C., followed by 30 cycles of 10 seconds at 98° C., 30 seconds at 55° C.and 2 minutes at 72° C., followed by 10 minutes at 72° C. The resultingPCR fragment is analyzed on agarose gel, purified (QIAquick GelExtraction kit, Cat. Nr. 28706, Qiagen), blunt-end cloned into SrfI-linearized pGNA49A vector (reference to WO00188121A1), and sequenced.The sequence of the resulting PCR product corresponds to the respectivesequence as given in Table 8-TC. The recombinant vector harbouring thissequence is named pGBNJ00 XX.

E. Expression and Production of a Double-Stranded RNA Target in TwoStrains of Escherichia coli: (1) AB309-105, and, (2) BL21(DE3)

The procedures described below are followed in order to express suitablelevels of insect-active double-stranded RNA of insect target inbacteria. An RNaseIII-deficient strain, AB309-105, is used in comparisonto wild-type RNaseIII-containing bacteria, BL21 (DE3).

Transformation of AB309-105 and BL21(DE3)

Three hundred ng of the plasmid are added to and gently mixed in a 50 μlaliquot of ice-chilled chemically competent E. coli strain AB309-105 orBL21(DE3). The cells are incubated on ice for 20 minutes beforesubjecting them to a heat shock treatment of 37° C. for 5 minutes, afterwhich the cells are placed back on ice for a further 5 minutes. Fourhundred and fifty μl of room temperature SOC medium is added to thecells and the suspension incubated on a shaker (250 rpm) at 37° C. for 1hour. One hundred μl of the bacterial cell suspension is transferred toa 500 ml conical flask containing 150 ml of liquid Luria-Bertani (LB)broth supplemented with 100 μg/ml carbenicillin antibiotic. The cultureis incubated on an Innova 4430 shaker (250 rpm) at 37° C. overnight (16to 18 hours).

Chemical Induction of Double-Stranded RNA Expression in AB309-105 andBL21(DE3)

Expression of double-stranded RNA from the recombinant vector, pGBNJ003,in the bacterial strain AB309-105 or BL21(DE3) is made possible sinceall the genetic components for controlled expression are present. In thepresence of the chemical inducer isopropylthiogalactoside, or IPTG, theT7 polymerase will drive the transcription of the target sequence inboth antisense and sense directions since these are flanked byoppositely oriented T7 promoters.

The optical density at 600 nm of the overnight bacterial culture ismeasured using an appropriate spectrophotometer and adjusted to a valueof 1 by the addition of fresh LB broth. Fifty ml of this culture istransferred to a 50 ml Falcon tube and the culture then centrifuged at3000 g at 15° C. for 10 minutes. The supernatant is removed and thebacterial pellet resuspended in 50 ml of fresh S complete medium (SNCmedium plus 5 μg/ml cholesterol) supplemented with 100 μg/mlcarbenicillin and 1 mM IPTG. The bacteria are induced for 2 to 4 hoursat room temperature.

Heat Treatment of Bacteria

Bacteria are killed by heat treatment in order to minimise the risk ofcontamination of the artificial diet in the test plates. However, heattreatment of bacteria expressing double-stranded RNA is not aprerequisite for inducing toxicity towards the insects due to RNAinterference. The induced bacterial culture is centrifuged at 3000 g atroom temperature for 10 minutes, the supernatant discarded and thepellet subjected to 80° C. for 20 minutes in a water bath. After heattreatment, the bacterial pellet is resuspended in 1.5 ml MilliQ waterand the suspension transferred to a microfuge tube. Several tubes areprepared and used in the bioassays for each refreshment. The tubes arestored at −20° C. until further use.

F. Laboratory Trials to Test Escherichia coli Expressing dsRNA TargetsAgainst Tribolium castaneum Plant-Based Bioassays

Whole plants are sprayed with suspensions of chemically induced bacteriaexpressing dsRNA prior to feeding the plants to RFB. The are grown fromin a plant growth room chamber. The plants are caged by placing a 500 mlplastic bottle upside down over the plant with the neck of the bottlefirmly placed in the soil in a pot and the base cut open and coveredwith a fine nylon mesh to permit aeration, reduce condensation insideand prevent insect escape. RFB are placed on each treated plant in thecage. Plants are treated with a suspension of E. coli AB309-105harbouring the pGBNJ001 plasmids or pGN29 plasmid. Different quantitiesof bacteria are applied to the plants: for instance 66, 22, and 7 units,where one unit is defined as 10⁹ bacterial cells in 1 ml of a bacterialsuspension at optical density value of 1 at 600 nm wavelength. In eachcase, a total volume of between 1 and 10 ml s sprayed on the plant withthe aid of a vaporizer. One plant is used per treatment in this trial.The number of survivors are counted and the weight of each survivorrecorded.

Spraying plants with a suspension of E. coli bacterial strain AB309-105expressing target dsRNA from pGBNJ003 lead to a dramatic increase ininsect mortality when compared to pGN29 control. These experiments showthat double-stranded RNA corresponding to an insect gene target sequenceproduced in either wild-type or RNaseIII-deficient bacterial expressionsystems is toxic towards the insect in terms of substantial increases ininsect mortality and growth/development delay for larval survivors. Itis also clear from these experiments that an exemplification is providedfor the effective protection of plants/crops from insect damage by theuse of a spray of a formulation consisting of bacteria expressingdouble-stranded RNA corresponding to an insect gene target.

Example 10 Myzus persicae (Green Peach Aphid) A. Cloning Myzus persicaePartial Sequences

High quality, intact RNA was isolated from nymphs of Myzus persicae(green peach aphid; source: Dr. Rachel Down, Insect & PathogenInteractions, Central Science Laboratory, Sand Hutton, York, YO41 1LZ,UK) using TRIzol Reagent (Cat. Nr. 15596-026/15596-018, Invitrogen,Rockville, Md., USA) following the manufacturer's instructions. GenomicDNA present in the RNA preparation was removed by DNase treatmentfollowing the manufacturer's instructions (Cat. Nr. 1700, Promega). cDNAwas generated using a commercially available kit (SuperScript™ IIIReverse Transcriptase, Cat. Nr. 18080044, Invitrogen, Rockville, Md.,USA) following the manufacturer's instructions.

To isolate cDNA sequences comprising a portion of the MP001, MP002,MP100, MP016 and MP027 genes, a series of PCR reactions with degenerateprimers were performed using Amplitaq Gold (Cat. Nr. N8080240, AppliedBiosystems) following the manufacturer's instructions.

The sequences of the degenerate primers used for amplification of eachof the genes are given in Table 2-MP. These primers were used inrespective PCR reactions with the following conditions: for MP001, MP002and MP016, 10 minutes at 95° C., followed by 40 cycles of 30 seconds at95° C., 1 minute at 50° C. and 1 minute 30 seconds at 72° C., followedby 7 minutes at 72° C.; for MP027, a touchdown program was used: 10minutes at 95° C., followed by 10 cycles of 30 seconds at 95° C., 40seconds at 60° C. with a decrease in temperature of 1° C. per cycle and1 minute 10 seconds at 72° C., followed by 30 cycles of 30 seconds at95° C., 40 seconds at 50° C. and 1 minute 10 seconds at 72° C., followedby 7 minutes at 72° C.; for MP010, 10 minutes at 95° C., followed by 40cycles of 30 seconds at 95° C., 1 minute at 54° C. and 3 minutes at 72°C., followed by 7 minutes at 72° C. The resulting PCR fragments wereanalyzed on agarose gel, purified (QIAquick Gel Extraction kit, Cat. Nr.28706, Qiagen), cloned into the pCR8/GW/TOPO vector (Cat. Nr. K2500-20,Invitrogen), and sequenced. The sequences of the resulting PCR productsare represented by the respective SEQ ID NO:s as given in Table 2-MP andare referred to as the partial sequences. The corresponding partialamino acid sequences are represented by the respective SEQ ID NO:s asgiven in Table 3-MP.

B. dsRNA Production of Myzus persicae Genes

dsRNA was synthesized in milligram amounts using the commerciallyavailable kit T7 Ribomax™ Express RNAi System (Cat. Nr. P1700, Promega).First two separate single 5′ T7 RNA polymerase promoter templates weregenerated in two separate PCR reactions, each reaction containing thetarget sequence in a different orientation relative to the T7 promoter.

For each of the target genes, the sense T7 template was generated usingspecific T7 forward and specific reverse primers. The sequences of therespective primers for amplifying the sense template for each of thetarget genes are given in Table 8-MP. A touchdown PCR was performed asfollows: 1 minute at 95° C., followed by 20 cycles of 30 seconds at 95°C., 30 seconds at 55° C. (for MP001, MP002, MP016, MP027 and gfp) or 30seconds at 50° C. (for MP010) with a decrease in temperature of 0.5° C.per cycle and 1 minute at 72° C., followed by 15 cycles of 30 seconds at95° C., 30 seconds at 45° C. and 1 minute at 72° C. followed by 10minutes at 72° C. The anti-sense T7 template was generated usingspecific forward and specific T7 reverse primers in a PCR reaction withthe same conditions as described above. The sequences of the respectiveprimers for amplifying the anti-sense template for each of the targetgenes are given in Table 8-MP. The resulting PCR products were analyzedon agarose gel and purified by PCR purification kit (Qiaquick PCRPurification Kit, Cat. Nr. 28106, Qiagen) and NaClO₄ precipitation. Thegenerated T7 forward and reverse templates were mixed to be transcribedand the resulting RNA strands were annealed, DNase and RNase treated,and purified by sodium acetate, following the manufacturer'sinstructions. The sense strand of the resulting dsRNA for each of thetarget genes is given in Table 8-MP.

C. Laboratory Trials to Test dsRNA Targets Using Liquid Artificial Dietfor Activity Against Myzus persicae

Liquid artificial diet for the green peach aphid, Myzus persicae, wasprepared based on the diet suitable for pea aphids (Acyrthosiphonpisum), as described by Febvay et al. (1988) [Influence of the aminoacid balance on the improvement of an artificial diet for a biotype ofAcyrthosiphon pisum (Homoptera: Aphididae). Can. J. Zool. 66:2449-2453], but with some modifications. The amino acids component ofthe diet was prepared as follows: in mg/100 ml, alanine 178.71,beta-alanine 6.22, arginine 244.9, asparagine 298.55, aspartic acid88.25, cysteine 29.59, glutamic acid 149.36, glutamine 445.61, glycine166.56, histidine 136.02, isoleucine 164.75, leucine 231.56, lysinehydrochloride 351.09, methionine 72.35, ornithine (HCl) 9.41,phenylalanine 293, proline 129.33, serine 124.28, threonine 127.16,tryptophane 42.75, tyrosine 38.63, L-valine 190.85. The amino acids weredissolved in 30 ml Milli-Q H₂O except for tyrosine which was firstdissolved in a few drops of 1 M HCl before adding to the amino acid mix.The vitamin mix component of the diet was prepared as a 5× concentratestock as follows: in mg/L, amino benzoic acid 100, ascorbic acid 1000,biotin 1, calcium panthothenate 50, choline chloride 500, folic acid 10,myoinositol 420, nicotinic acid 100, pyridoxine hydrochloride 25,riboflavin 5, thiamine hydrochloride 25. The riboflavin was dissolved in1 ml H₂O at 50° C. and then added to the vitamin mix stock. The vitaminmix was aliquoted in 20 ml per aliquot and stored at −20° C. One aliquotof vitamin mix was added to the amino acid solution. Sucrose andMgSO₄.7H₂O was added with the following amounts to the mix: 20 g and 242mg, respectively. Trace metal stock solution was prepared as follows: inmg/100 ml, CuSO₄.5H₂O 4.7, FeCl₃.6H₂O 44.5, MnCl₂.4H2O 6.5, NaCl 25.4,ZnCl₂ 8.3. Ten ml of the trace metal solution and 250 mg KH₂PO₄ wasadded to the diet and Milli-Q water was added to a final liquid dietvolume of 100 ml. The pH of the diet was adjusted to 7 with 1 M KOHsolution. The liquid diet was filter-sterilised through an 0.22 μmfilter disc (Millipore).

Green peach aphids (Myzus persicae; source: Dr. Rachel Down, Insect &Pathogen Interactions, Central Science Laboratory, Sand Hutton, York,YO41 1LZ, UK) were reared on 4- to 6-week-old oilseed rape (Brassicanapus variety SW Oban; source: Nick Balaam, Sw Seed Ltd., 49 North Road,Abington, Cambridge, CB 1 6AS, UK) in aluminium-framed cages containing70 μm mesh in a controlled environment chamber with the followingconditions: 23±2° C. and 60±5% relative humidity, with a 16:8 hourslight:dark photoperiod.

One day prior to the start of the bioassay, adults were collected fromthe rearing cages and placed on fresh detached oilseed rape leaves in aPetri dish and left overnight in the insect chamber. The following day,first-instar nymphs were picked and transferred to feeding chambers. Afeeding chamber comprised of 10 first instar nymphs placed in a smallPetri dish (with diameter 3 cm) covered with a single layer of thinlystretched parafilm M onto which 50 μl of diet was added. The chamber wassealed with a second layer of parafilm and incubated under the sameconditions as the adult cultures. Diet with dsRNA was refreshed everyother day and the insects' survival assessed on day 8 i.e. 8^(th) daypost bioassay start. Per treatment, 5 bioassay feeding chambers(replicates) were set up simultaneously. Test and control (gfp) dsRNAsolutions were incorporated into the diet to a final concentration of 2μg/μl. The feeding chambers were kept at 23±2° C. and 60±5% relativehumidity, with a 16:8 hours light:dark photoperiod. A Mann-Whitney testwas determined by GraphPad Prism version 4 to establish whether themedians do differ significantly between target 27 (MP027) and gfp dsRNA.

In the bioassay, feeding liquid artificial diet supplemented with intactnaked dsRNA from target 27 (SEQ ID NO: 1061) to nymphs of Myzus persicaeusing a feeding chamber, resulted in a significant increase inmortality, as shown in FIG. 1. Average percentage survivors for target27, gfp dsRNA and diet only treatment were 2, 34 and 82, respectively.Comparison of target 027 with gfp dsRNA groups using the Mann-Whitneytest resulted in an one-tailed P-value of 0.004 which indicates that themedian of target 027 is significantly different (P<0.05) from theexpected larger median of gfp dsRNA. The green peach aphids on theliquid diet with incorporated target 27 dsRNA were noticeably smallerthan those that were fed on diet only or with gfp dsRNA control (datanot presented).

D. Cloning of a GPA Gene Fragment in a Vector Suitable for BacterialProduction of Insect-Active Double-Stranded RNA

What follows is an example of cloning a DNA fragment corresponding to aGPA gene target in a vector for the expression of double-stranded RNA ina bacterial host, although any vector comprising a T7 promoter or anyother promoter for efficient transcription in bacteria, may be used(reference to WO0001846).

The sequences of the specific primers used for the amplification oftarget genes are provided in Table 8-MP. The template used is thepCR8/GW/topo vector containing any of target sequences. The primers areused in a PCR reaction with the following conditions: 5 minutes at 98°C., followed by 30 cycles of 10 seconds at 98° C., 30 seconds at 55° C.and 2 minutes at 72° C., followed by 10 minutes at 72° C. The resultingPCR fragment is analyzed on agarose gel, purified (QIAquick GelExtraction kit, Cat. Nr. 28706, Qiagen), blunt-end cloned into SrfI-linearized pGNA49A vector (reference to WO00188121A1), and sequenced.The sequence of the resulting PCR product corresponds to the respectivesequence as given in Table 8-MP. The recombinant vector harbouring thissequence is named pGBNJ00XX.

E. Expression and Production of a Double-Stranded RNA Target in TwoStrains of Escherichia coli: (1) AB309-105, and, (2) BL21(DE3)

The procedures described below are followed in order to express suitablelevels of insect-active double-stranded RNA of insect target inbacteria. An RNaseIII-deficient strain, AB309-105, is used in comparisonto wild-type RNaseIII-containing bacteria, BL21 (DE3).

Transformation of AB309-105 and BL21(DE3)

Three hundred ng of the plasmid are added to and gently mixed in a 50 μlaliquot of ice-chilled chemically competent E. coli strain AB309-105 orBL21(DE3). The cells are incubated on ice for 20 minutes beforesubjecting them to a heat shock treatment of 37° C. for 5 minutes, afterwhich the cells are placed back on ice for a further 5 minutes. Fourhundred and fifty μl of room temperature SOC medium is added to thecells and the suspension incubated on a shaker (250 rpm) at 37° C. for 1hour. One hundred μl of the bacterial cell suspension is transferred toa 500 ml conical flask containing 150 ml of liquid Luria-Bertani (LB)broth supplemented with 100 μg/ml carbenicillin antibiotic. The cultureis incubated on an Innova 4430 shaker (250 rpm) at 37° C. overnight (16to 18 hours).

Chemical Induction of Double-Stranded RNA Expression in AB309-105 andBL21(DE3)

Expression of double-stranded RNA from the recombinant vector, pGBNJ003,in the bacterial strain AB309-105 or BL21(DE3) is made possible sinceall the genetic components for controlled expression are present. In thepresence of the chemical inducer isopropylthiogalactoside, or IPTG, theT7 polymerase will drive the transcription of the target sequence inboth antisense and sense directions since these are flanked byoppositely oriented T7 promoters.

The optical density at 600 nm of the overnight bacterial culture ismeasured using an appropriate spectrophotometer and adjusted to a valueof 1 by the addition of fresh LB broth. Fifty ml of this culture istransferred to a 50 ml Falcon tube and the culture then centrifuged at3000 g at 15° C. for 10 minutes. The supernatant is removed and thebacterial pellet resuspended in 50 ml of fresh S complete medium (SNCmedium plus 5 μg/ml cholesterol) supplemented with 100 μg/mlcarbenicillin and 1 mM IPTG. The bacteria are induced for 2 to 4 hoursat room temperature.

Heat Treatment of Bacteria

Bacteria are killed by heat treatment in order to minimise the risk ofcontamination of the artificial diet in the test plates. However, heattreatment of bacteria expressing double-stranded RNA is not aprerequisite for inducing toxicity towards the insects due to RNAinterference. The induced bacterial culture is centrifuged at 3000 g atroom temperature for 10 minutes, the supernatant discarded and thepellet subjected to 80° C. for 20 minutes in a water bath. After heattreatment, the bacterial pellet is resuspended in 1.5 ml MilliQ waterand the suspension transferred to a microfuge tube. Several tubes areprepared and used in the bioassays for each refreshment. The tubes arestored at −20° C. until further use.

F. Laboratory Trials to Test Escherichia coli Expressing dsRNA TargetsAgainst Myzus persicae Plant-Based Bioassays

Whole plants are sprayed with suspensions of chemically induced bacteriaexpressing dsRNA prior to feeding the plants to GPA. The are grown fromin a plant growth room chamber. The plants are caged by placing a 500 mlplastic bottle upside down over the plant with the neck of the bottlefirmly placed in the soil in a pot and the base cut open and coveredwith a fine nylon mesh to permit aeration, reduce condensation insideand prevent insect escape. GPA are placed on each treated plant in thecage. Plants are treated with a suspension of E. coli AB309-105harbouring the pGBNJ001 plasmids or pGN29 plasmid. Different quantitiesof bacteria are applied to the plants: for instance 66, 22, and 7 units,where one unit is defined as 109 bacterial cells in 1 ml of a bacterialsuspension at optical density value of 1 at 600 nm wavelength. In eachcase, a total volume of between 1 and 10 ml s sprayed on the plant withthe aid of a vaporizer. One plant is used per treatment in this trial.The number of survivors are counted and the weight of each survivorrecorded.

Spraying plants with a suspension of E. coli bacterial strain AB309-105expressing target dsRNA from pGBNJ03 lead to a dramatic increase ininsect mortality when compared to pGN29 control. These experiments showthat double-stranded RNA corresponding to an insect gene target sequenceproduced in either wild-type or RNaseIII-deficient bacterial expressionsystems is toxic towards the insect in terms of substantial increases ininsect mortality and growth/development delay for larval survivors. Itis also clear from these experiments that an exemplification is providedfor the effective protection of plants/crops from insect damage by theuse of a spray of a formulation consisting of bacteria expressingdouble-stranded RNA corresponding to an insect gene target.

Example 11 Nilaparvata lumens (Brown Plant Hopper) A. CloningNilaparvata lugens Partial Sequences

From high quality total RNA of Nilaparvata lugens (source: Dr. J. A.Gatehouse, Dept. Biological Sciences, Durham University, UK) cDNA wasgenerated using a commercially available kit (SuperScript™ III ReverseTranscriptase, Cat N^(o). 18080044, Invitrogen, Rockville, Md., USA)following the manufacturer's protocol.

To isolate cDNA sequences comprising a portion of the Nilaparvata lugensNL001, NL002, NL003, NL004, NL005, NL006, NL007, NL008, NL009, NL010,NL011, NL012, NL013, NL014, NL015, NL016, NL018, NL019, NL021, NL022,and NL027 genes, a series of PCR reactions with degenerate primers wereperformed using Amplitaq Gold (Cat N^(o). N8080240; Applied Biosystems)following the manufacturer's protocol.

The sequences of the degenerate primers used for amplification of eachof the genes are given in Table 2-NL. These primers were used inrespective PCR reactions with the following conditions: for NL001: 5minutes at 95° C., followed by 40 cycles of 30 seconds at 95° C., 1minute at 55° C. and 1 minute at 72° C., followed by 10 minutes at 72°C.: for NL002: 3 minutes at 95° C., followed by 40 cycles of 30 secondsat 95° C., 1 minute at 55° C. and 1 minute at 72° C., followed by 10minutes at 72° C.; for NL003: 3 minutes at 95° C., followed by 40 cyclesof 30 seconds at 95° C., 1 minute at 61° C. and 1 minute at 72° C.,followed by 10 minutes at 72° C.; for NL004: 10 minutes at 95° C.,followed by 40 cycles of 30 seconds at 95° C., 1 minute at 51° C. and 1minute at 72° C.; for NL005: 10 minutes at 95° C., followed by 40 cyclesof 30 seconds at 95° C., 1 minute at 54° C. and 1 minute at 72° C.,followed by 10 minutes at 72° C.; for NL006: 10 minutes at 95° C.,followed by 40 cycles of 30 seconds at 95° C., 1 minute at 55° C. and 3minute 30 seconds at 72° C., followed by 10 minutes at 72° C.; forNL007: 10 minutes at 95° C., followed by 40 cycles of 30 seconds at 95°C., 1 minute at 54° C. and 1 minute 15 seconds at 72° C., followed by 10minutes at 72° C.; for NL008: 10 minutes at 95° C., followed by 40cycles of 30 seconds at 95° C., 1 minute at 53° C. and 1 minute at 72°C., followed by 10 minutes at 72° C.; for NL009: 10 minutes at 95° C.,followed by 40 cycles of 30 seconds at 95° C., 1 minute at 55° C. and 1minute at 72° C., followed by 10 minutes at 72° C.; for NL010: 10minutes at 95° C., followed by 40 cycles of 30 seconds at 95° C., 1minute at 54° C. and 2 minute 30 seconds at 72° C., followed by 10minutes at 72° C.; for NL011: 10 minutes at 95° C., followed by 40cycles of 30 seconds at 95° C., 1 minute at 55° C. and 1 minute at 72°C.; for NL012: 10 minutes at 95° C., followed by 40 cycles of 30 secondsat 95° C., 1 minute at 55° C. and 1 minute at 72° C.; for NL013: 10minutes at 95° C., followed by 40 cycles of 30 seconds at 95° C., 1minute at 54° C. and 1 minute 10 seconds at 72° C., followed by 10minutes at 72° C.; for NL014: 10 minutes at 95° C., followed by 40cycles of 30 seconds at 95° C., 1 minute at 53° C. and 1 minute at 72°C., followed by 10 minutes at 72° C.; for NL015: 10 minutes at 95° C.,followed by 40 cycles of 30 seconds at 95° C., 1 minute at 54° C. and 1minute 40 seconds at 72° C., followed by 10 minutes at 72° C.; forNL016: 10 minutes at 95° C., followed by 40 cycles of 30 seconds at 95°C., 1 minute at 54° C. and 1 minute 40 seconds at 72° C., followed by 10minutes at 72° C.; for NL018: 10 minutes at 95° C., followed by 40cycles of 30 seconds at 95° C., 1 minute at 54° C. and 1 minute 35seconds at 72° C., followed by 10 minutes at 72° C.; for NL019: 10minutes at 95° C., followed by 40 cycles of 30 seconds at 95° C., 1minute at 55° C. and 1 minute at 72° C., followed by 10 minutes at 72°C.; for NL021: 10 minutes at 95° C., followed by 40 cycles of 30 secondsat 95° C., 1 minute at 54° C. and 1 minute 45 seconds at 72° C.,followed by 10 minutes at 72° C.: for NL022: 10 minutes at 95° C.,followed by 40 cycles of 30 seconds at 95° C., 1 minute at 54° C. and 1minute 45 seconds at 72° C., followed by 10 minutes at 72° C.; and forNL027: 10 minutes at 95° C., followed by 40 cycles of 30 seconds at 95°C., 1 minute at 54° C. and 1 minute 45 seconds at 72° C., followed by 10minutes at 72° C. The resulting PCR fragments were analyzed on agarosegel, purified (QIAquick Gel Extraction kit, Cat. Nr. 28706, Qiagen),cloned into the pCR8/GW/topo vector (Cat. Nr. K2500 20, Invitrogen), andsequenced. The sequences of the resulting PCR products are representedby the respective SEQ ID NO:s as given in Table 2-NL and are referred toas the partial sequences. The corresponding partial amino acid sequencesare represented by the respective SEQ ID NO:s as given in Table 3-NL.

B. Cloning of a Partial Sequence of the Nilaparvata lugens NL023 GeneVia EST Sequence

From high quality total RNA of Nilaparvata lugens (source: Dr. J. A.Gatehouse, Dept. Biological Sciences, Durham University, UK) cDNA wasgenerated using a commercially available kit (SuperScript™ III ReverseTranscriptase, Cat N^(o). 18080044, Invitrogen, Rockville, Md., USA)following the manufacturer's protocol.

A partial cDNA sequence, NL023, was amplified from Nilaparvata lugenscDNA which corresponded to a Nilaparvata lugens EST sequence in thepublic database Genbank with accession number CAH65679.2. To isolatecDNA sequences comprising a portion of the NL023 gene, a series of PCRreactions with EST based specific primers were performed usingPerfectShot™ ExTaq (Cat N^(o). RR005A, Takara Bio Inc.) following themanufacturer's protocol.

For NL023, the specific primers oGBKW002 and oGBKW003 (representedherein as SEQ ID NO: 1157 and SEQ ID NO: 1158, respectively) were usedin two independent PCR reactions with the following conditions: 3minutes at 95° C., followed by 30 cycles of 30 seconds at 95° C., 30seconds at 56° C. and 2 minutes at 72° C., followed by 10 minutes at 72°C. The resulting PCR products were analyzed on agarose gel, purified(QIAquick® Gel Extraction Kit; Cat. N^(o). 28706, Qiagen), cloned intothe pCR4-TOPO vector (Cat N^(o). K4575-40, Invitrogen) and sequenced.The consensus sequence resulting from the sequencing of both PCRproducts is herein represented by SEQ ID NO: 1111 and is referred to asthe partial sequence of the NL023 gene. The corresponding partial aminoacid sequence is herein represented as SEQ ID NO: 1112.

C. dsRNA Production of Nilaparvata lugens Genes

dsRNA was synthesized in milligram amounts using the commerciallyavailable kit T7 Ribomax™ Express RNAi System (Cat. Nr. P1700, Promega).First two separate single 5′ T7 RNA polymerase promoter templates weregenerated in two separate PCR reactions, each reaction containing thetarget sequence in a different orientation relative to the T7 promoter.

For each of the target genes, the sense T7 template was generated usingspecific T7 forward and specific reverse primers. The sequences of therespective primers for amplifying the sense template for each of thetarget genes are given in Table 4. The conditions in the PCR reactionswere as follows: for NL001: 4 minutes at 94° C., followed by 35 cyclesof 30 seconds at 94° C., 30 seconds at 60° C. and 1 minute at 72° C.,followed by 10 minutes at 72° C.; for NL002: 4 minutes at 94° C.,followed by 35 cycles of 30 seconds at 94° C., 30 seconds at 60° C. and1 minute at 72° C., followed by 10 minutes at 72° C.; for NL003: 4minutes at 94° C., followed by 35 cycles of 30 seconds at 94° C., 30seconds at 66° C. and 1 minute at 72° C., followed by 10 minutes at 72°C.; for NL004: 4 minutes at 95° C., followed by 35 cycles of 30 secondsat 95° C., 30 seconds at 54° C. and 1 minute at 72° C., followed by 10minutes at 72° C.; for NL005: 4 minutes at 95° C., followed by 35 cyclesof 30 seconds at 95° C., 30 seconds at 57° C. and 1 minute at 72° C.,followed by 10 minutes at 72° C.; for NL006: 4 minutes at 95° C.,followed by 35 cycles of 30 seconds at 95° C., 30 seconds at 54° C. and1 minute at 72° C., followed by 10 minutes at 72° C.; for NL007: 4minutes at 95° C., followed by 35 cycles of 30 seconds at 95° C., 30seconds at 51° C. and 1 minute at 72° C., followed by 10 minutes at 72°C.; for NL008: 4 minutes at 95° C., followed by 35 cycles of 30 secondsat 95° C., 30 seconds at 54° C. and 1 minute at 72° C., followed by 10minutes at 72° C.; for NL009: 4 minutes at 95° C., followed by 35 cyclesof 30 seconds at 95° C., 30 seconds at 54° C. and 1 minute at 72° C.,followed by 10 minutes at 72° C.; for NL010: 4 minutes at 95° C.,followed by 35 cycles of 30 seconds at 95° C., 30 seconds at 54° C. and1 minute at 72° C., followed by 10 minutes at 72° C.; for NL011: 4minutes at 95° C., followed by 35 cycles of 30 seconds at 95° C., 30seconds at 53° C. and 1 minute at 72° C., followed by 10 minutes at 72°C.; for NL012: 4 minutes at 95° C., followed by 35 cycles of 30 secondsat 95° C., 30 seconds at 53° C. and 1 minute at 72° C., followed by 10minutes at 72° C.; for NL013: 4 minutes at 95° C., followed by 35 cyclesof 30 seconds at 95° C., 30 seconds at 55° C. and 1 minute at 72° C.,followed by 10 minutes at 72° C.; for NL014: 4 minutes at 95° C.,followed by 35 cycles of 30 seconds at 95° C., 30 seconds at 51° C. and1 minute at 72° C., followed by 10 minutes at 72° C.; for NL015: 4minutes at 95° C., followed by 35 cycles of 30 seconds at 95° C., 30seconds at 55° C. and 1 minute at 72° C., followed by 10 minutes at 72°C.; for NL016: 4 minutes at 95° C., followed by 35 cycles of 30 secondsat 95° C., 30 seconds at 57° C. and 1 minute at 72° C., followed by 10minutes at 72° C.; for NL018: 4 minutes at 95° C., followed by 35 cyclesof 30 seconds at 95° C., 30 seconds at 55° C. and 1 minute at 72° C.,followed by 10 minutes at 72° C.; for NL019: 4 minutes at 95° C.,followed by 35 cycles of 30 seconds at 95° C., 30 seconds at 54° C. and1 minute at 72° C., followed by 10 minutes at 72° C.; for NL021: 4minutes at 95° C., followed by 35 cycles of 30 seconds at 95° C., 30seconds at 55° C. and 1 minute at 72° C., followed by 10 minutes at 72°C.; for NL022: 4 minutes at 95° C., followed by 35 cycles of 30 secondsat 95° C., 30 seconds at 53° C. and 1 minute at 72° C., followed by 10minutes at 72° C.; for NL023: 4 minutes at 95° C., followed by 35 cyclesof 30 seconds at 95° C., 30 seconds at 52° C. and 1 minute at 72° C.,followed by 10 minutes at 72° C.; and for NL027: 4 minutes at 95° C.,followed by 35 cycles of 30 seconds at 95° C., 30 seconds at 52° C. and1 minute at 72° C., followed by 10 minutes at 72° C. The anti-sense T7template was generated using specific forward and specific T7 reverseprimers in a PCR reaction with the same conditions as described above.The sequences of the respective primers for amplifying the anti-sensetemplate for each of the target genes are given in Table 4-NL. Theresulting PCR products were analyzed on agarose gel and purified by PCRpurification kit (Qiaquick PCR Purification Kit, Cat. Nr. 28106,Qiagen). The generated T7 forward and reverse templates were mixed to betranscribed and the resulting RNA strands were annealed, DNase and RNasetreated, and purified by sodium acetate, following the manufacturer'sinstructions, but with the following modification: RNA peppet is washedtwice in 70% ethanol. The sense strand of the resulting dsRNA for eachof the target genes is given in Table 8-NL.

The template DNA used for the PCR reactions with T7 primers on the greenfluorescent protein (gfp) control was the plasmid pPD96.12 (the FireLab, http://genome-www.stanford.edu/group/fire/), which contains thewild-type gfp coding sequence interspersed by 3 synthetic introns.Double-stranded RNA was synthesized using the commercially available kitT7 RiboMAX™ Express RNAi System (Cat. N^(o). P1700, Promega). First twoseparate single 5′ T7 RNA polymerase promoter templates were generatedin two separate PCR reactions, each reaction containing the targetsequence in a different orientation relative to the T7 promoter. Forgfp, the sense T7 template was generated using the specific T7 FW primeroGAU183 and the specific RV primer oGAU182 (represented herein as SEQ IDNO: 236 and SEQ ID NO: 237, respectively) in a PCR reaction with thefollowing conditions: 4 minutes at 95° C., followed by 35 cycles of 30seconds at 95° C., 30 seconds at 55° C. and 1 minute at 72° C., followedby 10 minutes at 72° C. The anti-sense T7 template was generated usingthe specific FW primer oGAU181 and the specific T7 RV primer oGAU184(represented herein as SEQ ID NO: 238 and SEQ ID NO: 239, respectively)in a PCR reaction with the same conditions as described above. Theresulting PCR products were analyzed on agarose gel and purified(QIAquick® PCR Purification Kit; Cat. N^(o). 28106, Qiagen). Thegenerated T7 FW and RV templates were mixed to be transcribed and theresulting RNA strands were annealed, DNase and RNase treated, andpurified by precipitation with sodium acetate and isopropanol, followingthe manufacturer's protocol, but with the following modification: RNApeppet is washed twice in 70% ethanol. The sense strands of theresulting dsRNA is herein represented by SEQ ID NO: 235.

D. Laboratory Trials to Screen dsRNA Targets Using Liquid ArtificialDiet for Activity Against Nilaparvata lugens

Liquid artificial diet (MMD-1) for the rice brown planthopper,Nilaparvata lugens, was prepared as described by Koyama (1988)[Artificial rearing and nutritional physiology of the planthoppers andleafhoppers (Homoptera: Delphacidae and Deltocephalidae) on a holidicdiet. JARQ 22: 20-27], but with a modification in final concentration ofdiet component sucrose: 14.4% (weight over volume) was used. Dietcomponents were prepared as separate concentrates: 10× mineral stock(stored at 4° C.), 2×amino acid stock (stored at −20° C.) and 10×vitamin stock (stored at −20° C.). The stock components were mixedimmediately prior to the start of a bioassay to 4/3× concentration toallow dilution with the test dsRNA solution (4×concentration), pHadjusted to 6.5, and filter-sterilised into approximately 500 μlaliquots.

Rice brown planthopper (Nilaparvata lugens) was reared on two-to-threemonth old rice (Oryza sativa cv Taichung Native 1) plants in acontrolled environment chamber: 27±2° C., 80% relative humidity, with a16:8 hours light:dark photoperiod. A feeding chamber comprised 10 firstor second instar nymphs placed in a small petri dish (with diameter 3cm) covered with a single layer of thinly stretched parafilm M ontowhich 50 μl of diet was added. The chamber was sealed with a secondlayer of parafilm and incubated under the same conditions as the adultcultures but with no direct light exposure. Diet with dsRNA wasrefreshed every other day and the insects' survival assessed daily. Pertreatment, 5 bioassay feeding chambers (replicates) were set upsimultaneously. Test and control (gfp) dsRNA solutions were incorporatedinto the diet to a final concentration of 2 mg/ml. The feeding chamberswere kept at 27±2° C., 80% relative humidity, with a 16:8 hourslight:dark photoperiod. Insect survival data were analysed using theKaplan-Meier survival curve model and the survival between groups werecompared using the logrank test (Prism version 4.0).

Feeding liquid artificial diet supplemented with intact naked dsRNAs toNilaparvata lugens in vitro using a feeding chamber resulted insignificant increases in nymphal mortalities as shown in four separatebioassays (FIGS. 1( a)-(d)-NL; Tables 1a-d-NL). These resultsdemonstrate that dsRNAs corresponding to different essential BPH genesshowed significant toxicity towards the rice brown planthopper.

Effect of gfp dsRNA on BPH survival in these bioassays is notsignificantly different to survival on diet only

Tables 10a-d-NL show a summary of the survival of Nilaparvata lugens onartificial diet supplemented with 2 mg/ml (final concentration) of thefollowing targets; in Table 10(a)-NL: NL002, NL003, NL005, NL010; inTable 10(b)-NL NL009, NL016; in Table 10(c)-NL NL014, NL018; and inTable 10(d)-NL NL013, NL015, NL021. In the survival analysis column, theeffect of RNAi is indicated as follows: +=significantly decreasedsurvival compared to gfp dsRNA control (alpha<0.05); −=no significantdifference in survival compared to gfp dsRNA control. Survival curveswere compared (between diet only and diet supplemented with test dsRNA,gfp dsRNA and test dsRNA, and diet only and gfp dsRNA) using the logranktest.

E. Laboratory Trials to Screen dsRNAs at Different Concentrations UsingArtificial Diet for Activity Against Nilaparvata lugens

Fifty μl of liquid artificial diet supplemented with differentconcentrations of target NL002 dsRNA, namely 1, 0.2, 0.08, and 0.04mg/ml (final concentration), was applied to the brown planthopperfeeding chambers. Diet with dsRNA was refreshed every other day and theinsects' survival assessed daily. Per treatment, 5 bioassay feedingchambers (replicates) were set up simultaneously. The feeding chamberswere kept at 27±2° C., 80% relative humidity, with a 16:8 hourslight:dark photoperiod. Insect survival data were analysed using theKaplan-Meier survival curve model and the survival between groups werecompared using the logrank test (Prism version 4.0).

Feeding liquid artificial diet supplemented with intact naked dsRNAs oftarget NL002 at different concentrations resulted in significantlyhigher BPH mortalities at final concentrations of as low as 0.04 mgdsRNA per ml diet when compared with survival on diet only, as shown inFIG. 2-NL and Table 9-NL. Table 9-NL summarizes the survival ofNilaparvata lugens artificial diet feeding trial supplemented with 1,0.2, 0.08, & 0.04 mg/ml (final concentration) of target NL002. In thesurvival analysis column the effect of RNAi is indicated as follows:+=significantly decreases survival compared to diet only control(alpha<0.05); −=no significant differences in survival compared to dietonly control. Survival curves were compared using the logrank test.

F. Cloning of a BPI Gene Fragment in a Vector Suitable for BacterialProduction of Insect-Active Double-Stranded RNA

What follows is an example of cloning a DNA fragment corresponding to aBPH gene target in a vector for the expression of double-stranded RNA ina bacterial host, although any vector comprising a T7 promoter or anyother promoter for efficient transcription in bacteria, may be used(reference to WO0001846).

The sequences of the specific primers used for the amplification oftarget genes are provided in Table 8. The template used is thepCR8/GW/topo vector containing any of target sequences. The primers areused in a PCR reaction with the following conditions: 5 minutes at 98°C., followed by 30 cycles of 10 seconds at 98° C., 30 seconds at 55° C.and 2 minutes at 72° C., followed by 10 minutes at 72° C. The resultingPCR fragment is analyzed on agarose gel, purified (QIAquick GelExtraction kit, Cat. Nr. 28706, Qiagen), blunt-end cloned into SrfI-linearized pGNA49A vector (reference to WO00188121A1), and sequenced.The sequence of the resulting PCR product corresponds to the respectivesequence as given in Table 8-NL. The recombinant vector harbouring thissequence is named pGBNJ00.

G. Expression and Production of a Double-Stranded RNA Target in TwoStrains of Escherichia coli: (1) AB309-105, and, (2) BL21(DE3)

The procedures described below are followed in order to express suitablelevels of insect-active double-stranded RNA of insect target inbacteria. An RNaseIII-deficient strain, AB309-105, is used in comparisonto wild-type RNaseIII-containing bacteria, BL21 (DE3).

Transformation of AB309-105 and BL21(DE3)

Three hundred ng of the plasmid are added to and gently mixed in a 50 μlaliquot of ice-chilled chemically competent E. coli strain AB309-105 orBL21(DE3). The cells are incubated on ice for 20 minutes beforesubjecting them to a heat shock treatment of 37° C. for 5 minutes, afterwhich the cells are placed back on ice for a further 5 minutes. Fourhundred and fifty μl of room temperature SOC medium is added to thecells and the suspension incubated on a shaker (250 rpm) at 37° C. for 1hour. One hundred μl of the bacterial cell suspension is transferred toa 500 ml conical flask containing 150 ml of liquid Luria-Bertani (LB)broth supplemented with 100 μg/ml carbenicillin antibiotic. The cultureis incubated on an Innova 4430 shaker (250 rpm) at 37° C. overnight (16to 18 hours).

Chemical Induction of Double-Stranded RNA Expression in AB309-105 andBL21(DE3)

Expression of double-stranded RNA from the recombinant vector, pGBNJO03,in the bacterial strain AB309-105 or BL21(DE3) is made possible sinceall the genetic components for controlled expression are present. In thepresence of the chemical inducer isopropylthiogalactoside, or IPTG, theT7 polymerase will drive the transcription of the target sequence inboth antisense and sense directions since these are flanked byoppositely oriented T7 promoters.

The optical density at 600 nm of the overnight bacterial culture ismeasured using an appropriate spectrophotometer and adjusted to a valueof 1 by the addition of fresh LB broth. Fifty ml of this culture istransferred to a 50 ml Falcon tube and the culture then centrifuged at3000 g at 15° C. for 10 minutes. The supernatant is removed and thebacterial pellet resuspended in 50 ml of fresh S complete medium (SNCmedium plus 5 μg/ml cholesterol) supplemented with 100 μg/mlcarbenicillin and 1 mM IPTG. The bacteria are induced for 2 to 4 hoursat room temperature.

Heat Treatment of Bacteria

Bacteria are killed by heat treatment in order to minimise the risk ofcontamination of the artificial diet in the test plates. However, heattreatment of bacteria expressing double-stranded RNA is not aprerequisite for inducing toxicity towards the insects due to RNAinterference. The induced bacterial culture is centrifuged at 3000 g atroom temperature for 10 minutes, the supernatant discarded and thepellet subjected to 80° C. for 20 minutes in a water bath. After heattreatment, the bacterial pellet is resuspended in 1.5 ml MilliQ waterand the suspension transferred to a microfuge tube. Several tubes areprepared and used in the bioassays for each refreshment. The tubes arestored at −20° C. until further use.

H. Laboratory Trials to Test Escherichia coli Expressing dsRNA TargetsAgainst Nilaparvata lugens Plant-Based Bioassays

Whole plants are sprayed with suspensions of chemically induced bacteriaexpressing dsRNA prior to feeding the plants to BPH. The are grown fromin a plant growth room chamber. The plants are caged by placing a 500 mlplastic bottle upside down over the plant with the neck of the bottlefirmly placed in the soil in a pot and the base cut open and coveredwith a fine nylon mesh to permit aeration, reduce condensation insideand prevent insect escape. BPH are placed on each treated plant in thecage. Plants are treated with a suspension of E. coli AB309-105harbouring the pGBNJ001 plasmids or pGN29 plasmid. Different quantitiesof bacteria are applied to the plants: for instance 66, 22, and 7 units,where one unit is defined as 10⁹ bacterial cells in 1 ml of a bacterialsuspension at optical density value of 1 at 600 nm wavelength. In eachcase, a total volume of between 1 and 10 ml s sprayed on the plant withthe aid of a vaporizer. One plant is used per treatment in this trial.The number of survivors are counted and the weight of each survivorrecorded.

Spraying plants with a suspension of E. coli bacterial strain AB309-105expressing target dsRNA from pGBNJO03 lead to a dramatic increase ininsect mortality when compared to pGN29 control. These experiments showthat double-stranded RNA corresponding to an insect gene target sequenceproduced in either wild-type or RNaseIII-deficient bacterial expressionsystems is toxic towards the insect in terms of substantial increases ininsect mortality and growth/development delay for larval survivors. Itis also clear from these experiments that an exemplification is providedfor the effective protection of plants/crops from insect damage by theuse of a spray of a formulation consisting of bacteria expressingdouble-stranded RNA corresponding to an insect gene target.

Example 10 Chilo suppressalis (Rice Striped Stem Borer) A. Cloning ofPartial Sequence of the Chilo suppressalis Genes Via Family PCR

High quality, intact RNA was isolated from the 4 different larval stagesof Chilo suppressalis (rice striped stem borer) using TRIzol Reagent(Cat. Nr. 15596-026/15596-018, Invitrogen, Rockville, Md., USA)following the manufacturer's instructions. Genomic DNA present in theRNA preparation was removed by DNase treatment following themanufacturer's instructions (Cat. Nr. 1700, Promega). cDNA was generatedusing a commercially available kit (SuperScript™ III ReverseTranscriptase, Cat. Nr. 18080044, Invitrogen, Rockville, Md., USA)following the manufacturer's instructions.

To isolate cDNA sequences comprising a portion of the CS001, CS002,CS003, CS006, CS007, CS009, CS011, CS013, CS014, CS015, CS016 and CS018genes, a series of PCR reactions with degenerate primers were performedusing Amplitaq Gold (Cat. Nr. N8080240, Applied Biosystems) followingthe manufacturer's instructions.

The sequences of the degenerate primers used for amplification of eachof the genes are given in Table 2-CS. These primers were used inrespective PCR reactions with the following conditions: 10 minutes at95° C., followed by 40 cycles of 30 seconds at 95° C., 1 minute at 55°C. and 1 minute at 72° C., followed by 10 minutes at 72° C. Theresulting PCR fragments were analyzed on agarose gel, purified (QIAquickGel Extraction kit, Cat. Nr. 28706, Qiagen), cloned into the pCR4/TOPOvector (Cat. Nr. K2500-20, Invitrogen), and sequenced. The sequences ofthe resulting PCR products are represented by the respective SEQ ID NO:sas given in Table 2-CS and are referred to as the partial sequences. Thecorresponding partial amino acid sequences are represented by therespective SEQ ID NO:s as given in Table 3-CS.

B. dsRNA Production of the Chilo suppressalis Genes

dsRNA was synthesized in milligram amounts using the commerciallyavailable kit T7 Ribomax™ Express RNAi System (Cat. Nr. P1700, Promega).First two separate single 5′ T7 RNA polymerase promoter templates weregenerated in two separate PCR reactions, each reaction containing thetarget sequence in a different orientation relative to the T7 promoter.

For each of the target genes, the sense T7 template was generated usingspecific T7 forward and specific reverse primers. The sequences of therespective primers for amplifying the sense template for each of thetarget genes are given in Table 8-CS. The conditions in the PCRreactions were as follows: 4 minutes at 95° C., followed by 35 cycles of30 seconds at 95° C., 30 seconds at 55° C. and 1 minute at 72° C.,followed by 10 minutes at 72° C. The anti-sense T7 template wasgenerated using specific forward and specific T7 reverse primers in aPCR reaction with the same conditions as described above. The sequencesof the respective primers for amplifying the anti-sense template foreach of the target genes are given in Table 8-CS. The resulting PCRproducts were analyzed on agarose gel and purified by PCR purificationkit (Qiaquick PCR Purification Kit, Cat. Nr. 28106, Qiagen) and NaClO₄precipitation. The generated T7 forward and reverse templates were mixedto be transcribed and the resulting RNA strands were annealed, DNase andRNase treated, and purified by sodium acetate, following themanufacturer's instructions. The sense strand of the resulting dsRNA foreach of the target genes is given in Table 8-CS.

C. Laboratory Trials to Test dsRNA Targets, Using Artificial Diet forActivity Against Chilo suppressalis Larvae

Rice striped stem borers, Chilo suppressalis, (origin: Syngenta, Stein,Switzerland) were maintained on a modified artificial diet based on thatdescribed by Kamano and Sato, 1985 (in: Handbook of Insect Rearing.Volumes I & II. P Singh and RF Moore, eds., Elsevier Science Publishers,Amsterdam and New York, 1985, pp 448). Briefly, a litre diet was made upas follows: 20 g of agar added to 980 ml of Milli-Q water andautoclaved; the agar solution was cooled down to approximately 55° C.and the remaining ingredients were added and mixed thoroughly: 40 g cornflour (Polenta), 20 g cellulose, 30 g sucrose, 30 g casein, 20 g wheatgerm (toasted), 8 g Wesson salt mixture, 12 g Vanderzant vitamin mix,1.8 g sorbic acid, 1.6 g nipagin (methylparaben), 0.3 g aureomycin, 0.4g cholesterol and 0.6 g L-cysteine. The diet was cooled down to approx.45° C. and poured into rearing trays or cups. The diet was left to setin a horizontal laminair flow cabin. Rice leaf sections with ovipositedeggs were removed from a cage housing adult moths and pinned to thesolid diet in the rearing cup or tray. Eggs were left to hatch andneonate larvae were available for bioassays and the maintenance of theinsect cultures. During the trials and rearings, the conditions were28±2° C. and 80±5% relative humidity, with a 16:8 hour light:darkphotoperiod.

The same artificial diet is used for the bioassays but in this case thediet is poured equally in 24 multiwell plates, with each well containing1 ml diet. Once the diet is set, the test formulations are applied tothe diet's surface (2 cm²), at the rate of 50 μl of 1 μg/μl dsRNA oftarget. The dsRNA solutions are left to dry and two first instar mothlarvae are placed in each well. After 7 days, the larvae are transferredto fresh treated diet in multiwell plates. At day 14 (i.e. 14 days postbioassay start) the number of live and dead insects is recorded andexamined for abnormalities. Twenty-four larvae in total are tested pertreatment.

An alternative bioassay is performed in which treated rice leaves arefed to neonate larvae of the rice striped stem borer. Small leafsections of Indica rice variety Taichung native 1 are dipped in 0.05%Triton X-100 solution containing 1 μg/μl of target dsRNA, left to dryand each section placed in a well of a 24 multiwell plate containinggellified 2% agar. Two neonates are transferred from the rearing tray toeach dsRNA treated leaf section (24 larvae per treatment). After 4 and 8days, the larvae are transferred to fresh treated rice leaf sections.The number of live and dead larvae are assessed on days 4, 8 and 12; anyabnormalities are also recorded.

D. Cloning of a SSB Gene Fragment in a Vector Suitable for BacterialProduction of Insect-Active Double-Stranded RNA

What follows is an example of cloning a DNA fragment corresponding to anSSB gene target in a vector for the expression of double-stranded RNA ina bacterial host, although any vector comprising a T7 promoter or anyother promoter for efficient transcription in bacteria, may be used(reference to WO0001846).

The sequences of the specific primers used for the amplification oftarget genes are provided in Table 8. The template used is thepCR8/GW/topo vector containing any of target sequences. The primers areused in a PCR reaction with the following conditions: 5 minutes at 98°C., followed by 30 cycles of 10 seconds at 98° C., 30 seconds at 55° C.and 2 minutes at 72° C., followed by 10 minutes at 72° C. The resultingPCR fragment is analyzed on agarose gel, purified (QIAquick GelExtraction kit, Cat. Nr. 28706, Qiagen), blunt-end cloned into SrfI-linearized pGNA49A vector (reference to WO00188121A1), and sequenced.The sequence of the resulting PCR product corresponds to the respectivesequence as given in Table 8-CS. The recombinant vector harbouring thissequence is named pGBNJ00XX.

E. Expression and Production of a Double-Stranded RNA Target in TwoStrains of Escherichia coli: (1) AB309-105, and, (2) BL21(DE3)

The procedures described below are followed in order to express suitablelevels of insect-active double-stranded RNA of insect target inbacteria. An RNaseIII-deficient strain, AB309-105, is used in comparisonto wild-type RNaseIII-containing bacteria, BL21 (DE3).

Transformation of AB309-105 and BL21(DE3)

Three hundred ng of the plasmid are added to and gently mixed in a 50 μlaliquot of ice-chilled chemically competent E. coli strain AB309-105 orBL21(DE3). The cells are incubated on ice for 20 minutes beforesubjecting them to a heat shock treatment of 37° C. for 5 minutes, afterwhich the cells are placed back on ice for a further 5 minutes. Fourhundred and fifty μl of room temperature SOC medium is added to thecells and the suspension incubated on a shaker (250 rpm) at 37° C. for 1hour. One hundred μl of the bacterial cell suspension is transferred toa 500 ml conical flask containing 150 ml of liquid Luria-Bertani (LB)broth supplemented with 100 μg/ml carbenicillin antibiotic. The cultureis incubated on an Innova 4430 shaker (250 rpm) at 37° C. overnight (16to 18 hours).

Chemical Induction of Double-Stranded RNA Expression in AB309-105 andBL21 (DE3)

Expression of double-stranded RNA from the recombinant vector, pGBNJ003,in the bacterial strain AB309-105 or BL21(DE3) is made possible sinceall the genetic components for controlled expression are present. In thepresence of the chemical inducer isopropylthiogalactoside, or IPTG, theT7 polymerase will drive the transcription of the target sequence inboth antisense and sense directions since these are flanked byoppositely oriented T7 promoters.

The optical density at 600 nm of the overnight bacterial culture ismeasured using an appropriate spectrophotometer and adjusted to a valueof 1 by the addition of fresh LB broth. Fifty ml of this culture istransferred to a 50 ml Falcon tube and the culture then centrifuged at3000 g at 15° C. for 10 minutes. The supernatant is removed and thebacterial pellet resuspended in 50 ml of fresh S complete medium (SNCmedium plus 5 μg/ml cholesterol) supplemented with 100 μg/mlcarbenicillin and 1 mM IPTG. The bacteria are induced for 2 to 4 hoursat room temperature.

Heat Treatment of Bacteria

Bacteria are killed by heat treatment in order to minimise the risk ofcontamination of the artificial diet in the test plates. However, heattreatment of bacteria expressing double-stranded RNA is not aprerequisite for inducing toxicity towards the insects due to RNAinterference. The induced bacterial culture is centrifuged at 3000 g atroom temperature for 10 minutes, the supernatant discarded and thepellet subjected to 80° C. for 20 minutes in a water bath. After heattreatment, the bacterial pellet is resuspended in 1.5 ml MilliQ waterand the suspension transferred to a microfuge tube. Several tubes areprepared and used in the bioassays for each refreshment. The tubes arestored at −20° C. until further use.

F. Laboratory Trials to Test Escherichia coli Expressing dsRNA TargetsAgainst Chilo suppressalis Plant-Based Bioassays

Whole plants are sprayed with suspensions of chemically induced bacteriaexpressing dsRNA prior to feeding the plants to SSB. The are grown fromin a plant growth room chamber. The plants are caged by placing a 500 mlplastic bottle upside down over the plant with the neck of the bottlefirmly placed in the soil in a pot and the base cut open and coveredwith a fine nylon mesh to permit aeration, reduce condensation insideand prevent insect escape. SSB are placed on each treated plant in thecage. Plants are treated with a suspension of E. coli AB309-105harbouring the pGBNJ001 plasmids or pGN29 plasmid. Different quantitiesof bacteria are applied to the plants: for instance 66, 22, and 7 units,where one unit is defined as 10⁹ bacterial cells in 1 ml of a bacterialsuspension at optical density value of 1 at 600 nm wavelength. In eachcase, a total volume of between 1 and 10 ml s sprayed on the plant withthe aid of a vaporizer. One plant is used per treatment in this trial.The number of survivors are counted and the weight of each survivorrecorded.

Spraying plants with a suspension of E. coli bacterial strain AB309-105expressing target dsRNA from pGBNJ003 lead to a dramatic increase ininsect mortality when compared to pGN29 control. These experiments showthat double-stranded RNA corresponding to an insect gene target sequenceproduced in either wild-type or RNaseIII-deficient bacterial expressionsystems is toxic towards the insect in terms of substantial increases ininsect mortality and growth/development delay for larval survivors. Itis also clear from these experiments that an exemplification is providedfor the effective protection of plants/crops from insect damage by theuse of a spray of a formulation consisting of bacteria expressingdouble-stranded RNA corresponding to an insect gene target.

Example 9 Plutella xylostella (Diamondback Moth) A. Cloning of a PartialSequence of the Plutella xylostella

High quality, intact RNA was isolated from all the different larvalstages of Plutella xylostella (Diamondback moth; source: Dr. LaraSenior, Insect Investigations Ltd., Capital Business Park, Wentloog,Cardiff, CF3 2PX, Wales, UK) using TRIzol Reagent (Cat, Nr.15596-026/15596-018, Invitrogen, Rockville, Md., USA) following themanufacturer's instructions. Genomic DNA present in the RNA preparationwas removed by DNase treatment following the manufacturer's instructions(Cat. Nr. 1700, Promega). cDNA was generated using a commerciallyavailable kit (SuperScript™ III Reverse Transcriptase, Cat. Nr.18080044, Invitrogen, Rockville, Md., USA) following the manufacturer'sinstructions.

To isolate cDNA sequences comprising a portion of the PX001, PX009,PX010, PX015, PX016 genes, a series of PCR reactions with degenerateprimers were performed using Amplitaq Gold (Cat. Nr. N8080240, AppliedBiosystems) following the manufacturer's instructions.

The sequences of the degenerate primers used for amplification of eachof the genes are given in Table 2-PX. These primers were used inrespective PCR reactions with the following conditions: 10 minutes at95° C., followed by 40 cycles of 30 seconds at 95° C., 1 minute at 50°C. and 1 minute and 30 seconds at 72° C., followed by 7 minutes at 72°C. (for PX001, PX009, PX015, PX016); 10 minutes at 95° C., followed by40 cycles of 30 seconds at 95° C., 1 minute at 54° C. and 2 minute and30 seconds at 72° C., followed by 7 minutes at 72° C. (for PX010). Theresulting PCR fragments were analyzed on agarose gel, purified (QIAquickGel Extraction kit, Cat. Nr. 28706, Qiagen), cloned into thepCR8/GW/TOPO vector (Cat. Nr. K2500-20, Invitrogen) and sequenced. Thesequences of the resulting PCR products are represented by therespective SEQ ID NO:s as given in Table 2-PX and are referred to as thepartial sequences. The corresponding partial amino acid sequence arerepresented by the respective SEQ ID NO:s as given in Table 3-PX.

B. dsRNA Production of the Plutella xylostella Genes

dsRNA was synthesized in milligram amounts using the commerciallyavailable kit T7 Ribomax™ Express RNAi System (Cat. Nr. P1700, Promega).First two separate single 5′ T7 RNA polymerase promoter templates weregenerated in two separate PCR reactions, each reaction containing thetarget sequence in a different orientation relative to the T7 promoter.

For each of the target genes, the sense T7 template was generated usingspecific T7 forward and specific reverse primers. The sequences of therespective primers for amplifying the sense template for each of thetarget genes are given in Table 8-PX. The conditions in the PCRreactions were as follows: 1 minute at 95° C., followed by 20 cycles of30 seconds at 95° C., 30 seconds at 60° C. (−0.5° C./cycle) and 1 minuteat 72° C., followed by 15 cycles of 30 seconds at 95° C., 30 seconds at50° C. and 1 minute at 72° C., followed by 10 minutes at 72° C. Theanti-sense T7 template was generated using specific forward and specificT7 reverse primers in a PCR reaction with the same conditions asdescribed above. The sequences of the respective primers for amplifyingthe anti-sense template for each of the target genes are given in Table8-PX. The resulting PCR products were analyzed on agarose gel andpurified by PCR purification kit (Qiaquick PCR Purification Kit, Cat.Nr. 28106, Qiagen) and NaClO₄ precipitation. The generated T7 forwardand reverse templates were mixed to be transcribed and the resulting RNAstrands were annealed, DNase and RNase treated, and purified by sodiumacetate, following the manufacturer's instructions. The sense strand ofthe resulting dsRNA for each of the target genes is given in Table 8-PX.

C. Laboratory Trials to Test dsRNA Targets, Using Artificial Diet forActivity Against Plutella xylostella Larvae

Diamond-back moths, Plutella xylostella, were maintained at InsectInvestigations Ltd. (origin: Newcastle University, Newcastle-upon-Tyne,UK). The insects were reared on cabbage leaves. First instar, mixed sexlarvae (approximately 1 day old) were selected for use in the trial.Insects were maintained in Eppendorf tubes (1.5 ml capacity).Commercially available Diamond-back moth diet (Bio-Serv, New Jersey,USA), prepared following the manufacturer's instructions, was placed inthe lid of each tube (0.25 ml capacity, 8 mm diameter). While stillliquid, the diet was smoother over to remove excess and produce an evensurface.

Once the diet has set the test formulations are applied to the diet'ssurface, at the rate of 25 μl undiluted formulation (1 μg/μl dsRNA oftargets) per replicate. The test formulations are allowed to dry and onefirst instar moth larva is placed in each tube. The larva is placed onthe surface of the diet in the lid and the tube carefully closed. Thetubes are stored upside down, on their lids such that each larva remainson the surface of the diet. Twice weekly the larvae are transferred tonew Eppendorf tubes with fresh diet. The insects are provided withtreated diet for the first two weeks of the trial and thereafter withuntreated diet.

Assessments are made twice weekly for a total of 38 days at which pointall larvae are dead. At each assessment the insects are assessed as liveor dead and examined for abnormalities. Forty single larva replicatesare performed for each of the treatments. During the trial the testconditions are 23 to 26° C. and 50 to 65% relative humidity, with a 16:8hour light:dark photoperiod.

D. Cloning of a DBM Gene Fragment in a Vector Suitable for BacterialProduction of Insect-Active Double-Stranded RNA

What follows is an example of cloning a DNA fragment corresponding to aDBM gene target in a vector for the expression of double-stranded RNA ina bacterial host, although any vector comprising a T7 promoter or anyother promoter for efficient transcription in bacteria, may be used(reference to WO0001846).

The sequences of the specific primers used for the amplification oftarget genes are provided in Table 8-PX. The template used is thepCR8/GW/topo vector containing any of target sequences. The primers areused in a PCR reaction with the following conditions: 5 minutes at 98°C., followed by 30 cycles of 10 seconds at 98° C., 30 seconds at 55° C.and 2 minutes at 72° C., followed by 10 minutes at 72° C. The resultingPCR fragment is analyzed on agarose gel, purified (QIAquick GelExtraction kit, Cat. Nr. 28706, Qiagen), blunt-end cloned into SrfI-linearized pGNA49A vector (reference to WO00188121A1), and sequenced.The sequence of the resulting PCR product corresponds to the respectivesequence as given in Table 8-PX. The recombinant vector harbouring thissequence is named pGBNJ00XX.

E. Expression and Production of a Double-Stranded RNA Target in TwoStrains of Escherichia coli: (1) AB309-105, and, (2) BL21(DE3)

The procedures described below are followed in order to express suitablelevels of insect-active double-stranded RNA of insect target inbacteria. An RNaseIII-deficient strain, AB309-105, is used in comparisonto wild-type RNaseIII-containing bacteria, BL21(DE3).

Transformation of AB309-105 and BL21(DE3)

Three hundred ng of the plasmid are added to and gently mixed in a 50 μlaliquot of ice-chilled chemically competent E. coli strain AB309-105 orBL21(DE3). The cells are incubated on ice for 20 minutes beforesubjecting them to a heat shock treatment of 37° C. for 5 minutes, afterwhich the cells are placed back on ice for a further 5 minutes. Fourhundred and fifty μl of room temperature SOC medium is added to thecells and the suspension incubated on a shaker (250 rpm) at 37° C. for 1hour. One hundred μl of the bacterial cell suspension is transferred toa 500 ml conical flask containing 150 ml of liquid Luria-Bertani (LB)broth supplemented with 100 μg/ml carbenicillin antibiotic. The cultureis incubated on an Innova 4430 shaker (250 rpm) at 37° C. overnight (16to 18 hours).

Chemical Induction of Double-Stranded RNA Expression in AB309-105 andBL21(DE3)

Expression of double-stranded RNA from the recombinant vector, pGBNJ003,in the bacterial strain AB309-105 or BL21(DE3) is made possible sinceall the genetic components for controlled expression are present. In thepresence of the chemical inducer isopropylthiogalactoside, or IPTG, theT7 polymerase will drive the transcription of the target sequence inboth antisense and sense directions since these are flanked byoppositely oriented T7 promoters.

The optical density at 600 nm of the overnight bacterial culture ismeasured using an appropriate spectrophotometer and adjusted to a valueof 1 by the addition of fresh LB broth. Fifty ml of this culture istransferred to a 50 ml Falcon tube and the culture then centrifuged at3000 g at 15° C. for 10 minutes. The supernatant is removed and thebacterial pellet resuspended in 50 ml of fresh S complete medium (SNCmedium plus 5 μg/ml cholesterol) supplemented with 100 μg/mlcarbenicillin and 1 mM IPTG. The bacteria are induced for 2 to 4 hoursat room temperature.

Heat Treatment of Bacteria

Bacteria are killed by heat treatment in order to minimise the risk ofcontamination of the artificial diet in the test plates. However, heattreatment of bacteria expressing double-stranded RNA is not aprerequisite for inducing toxicity towards the insects due to RNAinterference. The induced bacterial culture is centrifuged at 3000 g atroom temperature for 10 minutes, the supernatant discarded and thepellet subjected to 80° C. for 20 minutes in a water bath. After heattreatment, the bacterial pellet is resuspended in 1.5 ml MilliQ waterand the suspension transferred to a microfuge tube. Several tubes areprepared and used in the bioassays for each refreshment. The tubes arestored at −20° C. until further use.

F. Laboratory Trials to Test Escherichia coli Expressing dsRNA TargetsAgainst Plutella xylostella Plant-Based Bioassays

Whole plants are sprayed with suspensions of chemically induced bacteriaexpressing dsRNA prior to feeding the plants to DBM. The are grown fromin a plant growth room chamber. The plants are caged by placing a 500 mlplastic bottle upside down over the plant with the neck of the bottlefirmly placed in the soil in a pot and the base cut open and coveredwith a fine nylon mesh to permit aeration, reduce condensation insideand prevent insect escape. DBM are placed on each treated plant in thecage. Plants are treated with a suspension of E. coli AB309-105harbouring the pGBNJ001 plasmids or pGN29 plasmid. Different quantitiesof bacteria are applied to the plants: for instance 66, 22, and 7 units,where one unit is defined as 10⁹ bacterial cells in 1 ml of a bacterialsuspension at optical density value of 1 at 600 nm wavelength. In eachcase, a total volume of between 1 and 10 ml sprayed on the plant withthe aid of a vaporizer. One plant is used per treatment in this trial.The number of survivors are counted and the weight of each survivorrecorded.

Spraying plants with a suspension of E. coli bacterial strain AB309-105expressing target dsRNA from pGBNJ003 lead to a dramatic increase ininsect mortality when compared to pGN29 control. These experiments showthat double-stranded RNA corresponding to an insect gene target sequenceproduced in either wild-type or RNaseIII-deficient bacterial expressionsystems is toxic towards the insect in terms of substantial increases ininsect mortality and growth/development delay for larval survivors. Itis also clear from these experiments that an exemplification is providedfor the effective protection of plants/crops from insect damage by theuse of a spray of a formulation consisting of bacteria expressingdouble-stranded RNA corresponding to an insect gene target.

Example 12 Acheta domesticus (House Cricket) A. Cloning Achetadomesticus Partial Sequences

High quality, intact RNA was isolated from all the different insectstages of Acheta domesticus (house cricket; source: Dr. Lara Senior,Insect Investigations Ltd., Capital Business Park, Wentloog, Cardiff,CF3 2PX, Wales, UK) using TRIzol Reagent (Cat. Nr. 15596-026/15596-018,Invitrogen, Rockville, Md., USA) following the manufacturer'sinstructions. Genomic DNA present in the RNA preparation was removed byDNase treatment following the manufacturer's instructions (Cat. Nr.1700, Promega). cDNA was generated using a commercially available kit(SuperScript™ III Reverse Transcriptase, Cat. Nr. 18080044, Invitrogen,Rockville, Md., USA) following the manufacturer's instructions.

To isolate cDNA sequences comprising a portion of the AD001, AD002,AD009, AD015 and AD016 genes, a series of PCR reactions with degenerateprimers were performed using Amplitaq Gold (Cat. Nr. N8080240, AppliedBiosystems) following the manufacturer's instructions.

The sequences of the degenerate primers used for amplification of eachof the genes are given in Table 2-AD. These primers were used inrespective PCR reactions with the following conditions: 10 minutes at95° C., followed by 40 cycles of 30 seconds at 95° C., 1 minute at 50°C. and 1 minute and 30 seconds at 72° C., followed by 7 minutes at 72°C. The resulting PCR fragments were analyzed on agarose gel, purified(QIAquick Gel Extraction kit, Cat. Nr. 28706, Qiagen), cloned into thepCR8/GW/topo vector (Cat. Nr. K2500 20, Invitrogen) and sequenced. Thesequences of the resulting PCR products are represented by therespective SEQ ID NO:s as given in Table 2-AD and are referred to as thepartial sequences. The corresponding partial amino acid sequence arerepresented by the respective SEQ ID NO:s as given in Table 3-AD.

B. dsRNA Production of the Acheta domesticus Genes

dsRNA was synthesized in milligram amounts using the commerciallyavailable kit T7 Ribomax™ Express RNAi System (Cat. Nr. P1700, Promega).First two separate single 5′ T7 RNA polymerase promoter templates weregenerated in two separate PCR reactions, each reaction containing thetarget sequence in a different orientation relative to the T7 promoter.

For each of the target genes, the sense T7 template was generated usingspecific T7 forward and specific reverse primers. The sequences of therespective primers for amplifying the sense template for each of thetarget genes are given in Table 8-AD. The conditions in the PCRreactions were as follows: 1 minute at 95° C., followed by 20 cycles of30 seconds at 95° C., 30 seconds at 60° C. (−0.5° C./cycle) and 1 minuteat 72° C., followed by 15 cycles of 30 seconds at 95° C., 30 seconds at50° C. and 1 minute at 72° C., followed by 10 minutes at 72° C. Theanti-sense T7 template was generated using specific forward and specificT7 reverse primers in a PCR reaction with the same conditions asdescribed above. The sequences of the respective primers for amplifyingthe anti-sense template for each of the target genes are given in Table8-AD. The resulting PCR products were analyzed on agarose gel andpurified by PCR purification kit (Qiaquick PCR Purification Kit, Cat.Nr. 28106, Qiagen) and NaClO₄ precipitation. The generated T7 forwardand reverse templates were mixed to be transcribed and the resulting RNAstrands were annealed, DNase and RNase treated, and purified by sodiumacetate, following the manufacturer's instructions. The sense strand ofthe resulting dsRNA for each of the target genes is given in Table 8-AD.

C. Laboratory Trials to Test dsRNA Targets, Using Artificial Diet forActivity Against Acheta domesticus Larvae

House crickets, Acheta domesticus, were maintained at InsectInvestigations Ltd. (origin: Blades Biological Ltd., Kent, UK). Theinsects were reared on bran pellets and cabbage leaves. Mixed sex nymphsof equal size and no more than 5 days old were selected for use in thetrial. Double-stranded RNA is mixed with a wheat-based pelleted rodentdiet (rat and mouse standard diet, B & K Universal Ltd., Grimston,Aldbrough, Hull, UK). The diet, BK001P, contains the followingingredients in descending order by weight: wheat, soya, wheatfeed,barley, pellet binder, rodent 5 vit min, fat blend, dicalcium phosphate,mould carb. The pelleted rodent diet is finely ground and heat-treatedin a microwave oven prior to mixing, in order to inactivate any enzymecomponents. All rodent diet is taken from the same batch in order toensure consistency. The ground diet and dsRNA are mixed thoroughly andformed into small pellets of equal weight, which are allowed to dryovernight at room temperature.

Double-stranded RNA samples from targets and gfp control atconcentrations 10 μg/μl were applied in the ratio 1 g ground diet plus 1ml dsRNA solution, thereby resulting in an application rate of 10 mgdsRNA per g pellet. Pellets are replaced weekly. The insects areprovided with treated pellets for the first three weeks of the trial.Thereafter untreated pellets are provided. Insects are maintained withinlidded plastic containers (9 cm diameter, 4.5 cm deep), ten percontainer. Each arena contains one treated bait pellet and one watersource (damp cotton wool ball), each placed in a separate small weighboat. The water is replenished ad lib throughout the experiment.

Assessments are made at twice weekly intervals, with no more than fourdays between assessments, until all the control insects had either diedor moulted to the adult stage (84 days). At each assessment the insectsare assessed as live or dead, and examined for abnormalities. From day46 onwards, once moulting to adult has commenced, all insects (live anddead) are assessed as nymph or adult. Surviving insects are weighed onday 55 of the trial. Four replicates are performed for each of thetreatments. During the trial the test conditions are 25 to 33° C. and 20to 25% relative humidity, with a 12:12 hour light:dark photoperiod.

D. Cloning of a HC Gene Fragment in a Vector Suitable for BacterialProduction of Insect-Active Double-Stranded RNA

What follows is an example of cloning a DNA fragment corresponding to aHC gene target in a vector for the expression of double-stranded RNA ina bacterial host, although any vector comprising a T7 promoter or anyother promoter for efficient transcription in bacteria, may be used(reference to WO0001846).

The sequences of the specific primers used for the amplification oftarget genes are provided in Table 8. The template used is thepCR8/GW/topo vector containing any of target sequences. The primers areused in a PCR reaction with the following conditions: 5 minutes at 98°C., followed by 30 cycles of 10 seconds at 98° C., 30 seconds at 55° C.and 2 minutes at 72° C., followed by 10 minutes at 72° C. The resultingPCR fragment is analyzed on agarose gel, purified (QIAquick GelExtraction kit, Cat. Nr. 28706, Qiagen), blunt-end cloned into SrfI-linearized pGNA49A vector (reference to WO00188121A1), and sequenced.The sequence of the resulting PCR product corresponds to the respectivesequence as given in Table 8-AD. The recombinant vector harbouring thissequence is named pGBNJ00XX.

E. Expression and Production of a Double-Stranded RNA Target in TwoStrains of Escherichia coli: (1) AB309-105, and, (2) BL21(DE3)

The procedures described below are followed in order to express suitablelevels of insect-active double-stranded RNA of insect target inbacteria. An RNaseIII-deficient strain, AB309-105, is used in comparisonto wild-type RNaseIII-containing bacteria, BL21 (DE3).

Transformation of AB309-105 and BL21(DE3)

Three hundred ng of the plasmid are added to and gently mixed in a 50 μlaliquot of ice-chilled chemically competent E. coli strain AB309-105 orBL21(DE3). The cells are incubated on ice for 20 minutes beforesubjecting them to a heat shock treatment of 37° C. for 5 minutes, afterwhich the cells are placed back on ice for a further 5 minutes. Fourhundred and fifty μl of room temperature SOC medium is added to thecells and the suspension incubated on a shaker (250 rpm) at 37° C. for 1hour. One hundred μl of the bacterial cell suspension is transferred toa 500 ml conical flask containing 150 ml of liquid Luria-Bertani (LB)broth supplemented with 100 μg/ml carbenicillin antibiotic. The cultureis incubated on an Innova 4430 shaker (250 rpm) at 37° C. overnight (16to 18 hours).

Chemical Induction of Double-Stranded RNA Expression in AB309-105 andBL21 (DE3)

Expression of double-stranded RNA from the recombinant vector, pGBNJ003,in the bacterial strain AB309-105 or BL21(DE3) is made possible sinceall the genetic components for controlled expression are present. In thepresence of the chemical inducer isopropylthiogalactoside, or IPTG, theT7 polymerase will drive the transcription of the target sequence inboth antisense and sense directions since these are flanked byoppositely oriented T7 promoters.

The optical density at 600 nm of the overnight bacterial culture ismeasured using an appropriate spectrophotometer and adjusted to a valueof 1 by the addition of fresh LB broth. Fifty ml of this culture istransferred to a 50 ml Falcon tube and the culture then centrifuged at3000 g at 15° C. for 10 minutes. The supernatant is removed and thebacterial pellet resuspended in 50 ml of fresh S complete medium (SNCmedium plus 5 μg/ml cholesterol) supplemented with 100 μg/mlcarbenicillin and 1 mM IPTG. The bacteria are induced for 2 to 4 hoursat room temperature.

Heat Treatment of Bacteria

Bacteria are killed by heat treatment in order to minimise the risk ofcontamination of the artificial diet in the test plates. However, heattreatment of bacteria expressing double-stranded RNA is not aprerequisite for inducing toxicity towards the insects due to RNAinterference. The induced bacterial culture is centrifuged at 3000 g atroom temperature for 10 minutes, the supernatant discarded and thepellet subjected to 80° C. for 20 minutes in a water bath. After heattreatment, the bacterial pellet is resuspended in 1.5 ml MilliQ waterand the suspension transferred to a microfuge tube. Several tubes areprepared and used in the bioassays for each refreshment. The tubes arestored at −20° C. until further use.

F. Laboratory Trials to Test Escherichia coli Expressing dsRNA TargetsAgainst Acheta domesticus Plant-Based Bioassays

Whole plants are sprayed with suspensions of chemically induced bacteriaexpressing dsRNA prior to feeding the plants to HC. The are grown fromin a plant growth room chamber. The plants are caged by placing a 500 mlplastic bottle upside down over the plant with the neck of the bottlefirmly placed in the soil in a pot and the base cut open and coveredwith a fine nylon mesh to permit aeration, reduce condensation insideand prevent insect escape. HC are placed on each treated plant in thecage. Plants are treated with a suspension of E. coli AB309-105harbouring the pGBNJ001 plasmids or pGN29 plasmid. Different quantitiesof bacteria are applied to the plants: for instance 66, 22, and 7 units,where one unit is defined as 10⁹ bacterial cells in 1 ml of a bacterialsuspension at optical density value of 1 at 600 nm wavelength. In eachcase, a total volume of between 1 and 10 ml s sprayed on the plant withthe aid of a vaporizer. One plant is used per treatment in this trial.The number of survivors are counted and the weight of each survivorrecorded.

Spraying plants with a suspension of E. coli bacterial strain AB309-105expressing target dsRNA from pGBNJO03 lead to a dramatic increase ininsect mortality when compared to pGN29 control. These experiments showthat double-stranded RNA corresponding to an insect gene target sequenceproduced in either wild-type or RNaseIII-deficient bacterial expressionsystems is toxic towards the insect in terms of substantial increases ininsect mortality and growth/development delay for larval survivors. Itis also clear from these experiments that an exemplification is providedfor the effective protection of plants/crops from insect damage by theuse of a spray of a formulation consisting of bacteria expressingdouble-stranded RNA corresponding to an insect gene target.

Example 13 Pyricularia grisea (Rice Blast) A. Cloning P. grisea PartialSequences

High quality, intact RNA is isolated from different growth stages of P.grisea using TRIzol Reagent (Cat. Nr. 15596-026/15596-018, Invitrogen,Rockville, Md., USA) following the manufacturer's instructions. GenomicDNA present in the RNA preparation is removed by DNase treatmentfollowing the manufacturer's instructions (Cat. Nr. 1700, Promega). cDNAis generated using a commercially available kit (SuperScript™ IIIReverse Transcriptase, Cat. Nr. 18080044, Invitrogen, Rockville, Md.,USA) following the manufacturer's instructions.

To isolate cDNA sequences comprising a portion of a target gene, PCR isperformed with degenerate primers using Amplitaq Gold (Cat. Nr.N8080240, Applied Biosystems) following the manufacturer's instructions.The resultant PCR products are fractionated and sequenced.

B. dsRNA Production of P. grisea Genes

dsRNA is synthesized in milligram amounts using a commercially availablekit, such as T7 Ribomax™ Express RNAi System (Cat. Nr. P1700, Promega),following the manufacturer's instructions. The resulting PCR productsare analyzed on an agarose gel and purified by a PCR purification kit(e.g. Qiaquick PCR Purification Kit, Cat. Nr. 28106, Qiagen) and NaClO₄precipitation. The product T7 forward and reverse templates are mixedand the resulting RNA strands are annealed, then DNase and RNasetreated, and purified by sodium acetate, following the manufacturer'sinstructions.

C. Expression and Production of a Double-Stranded RNA Target in TwoStrains of Escherichia coli: (1) AB309-105, and, (2) BL21(DE3)

The procedures described below are followed in order to express suitablelevels of fungal double-stranded RNA of fungal target in bacteria. AnRNaseIII-deficient strain, AB309-105, is used in comparison to wild-typeRNaseIII-containing bacteria, BL21 (DE3).

Transformation of AB309-105 and BL21(DE3)

Three hundred ng of the plasmid are added to and gently mixed in a 50 μlaliquot of ice-chilled chemically competent E. coli strain AB309-105 orBL21(DE3). The cells are incubated on ice for 20 minutes beforesubjecting them to a heat shock treatment of 37° C. for 5 minutes, afterwhich the cells are placed back on ice for a further 5 minutes. Fourhundred and fifty μl of room temperature SOC medium is added to thecells and the suspension incubated on a shaker (250 rpm) at 37° C. for 1hour. One hundred μl of the bacterial cell suspension is transferred toa 500 ml conical flask containing 150 ml of liquid Luria-Bertani (LB)broth supplemented with 100 μg/ml carbenicillin antibiotic. The cultureis incubated on an Innova 4430 shaker (250 rpm) at 37° C. overnight (16to 18 hours).

Chemical Induction of Double-Stranded RNA Expression in AB309-105 andBL21 (DE3)

Expression of double-stranded RNA from the recombinant vector, pGBNJ003,in the bacterial strain AB309-105 or BL21(DE3) is made possible sinceall the genetic components for controlled expression are present. In thepresence of the chemical inducer isopropylthiogalactoside, or IPTG, theT7 polymerase will drive the transcription of the target sequence inboth antisense and sense directions since these are flanked byoppositely oriented T7 promoters.

The optical density at 600 nm of the overnight bacterial culture ismeasured using an appropriate spectrophotometer and adjusted to a valueof 1 by the addition of fresh LB broth. Fifty ml of this culture istransferred to a 50 ml Falcon tube and the culture then centrifuged at3000 g at 15° C. for 10 minutes. The supernatant is removed and thebacterial pellet resuspended in 50 ml of fresh S complete medium (SNCmedium plus 5 μg/ml cholesterol) supplemented with 100 μg/mlcarbenicillin and 1 mM IPTG. The bacteria are induced for 2 to 4 hoursat room temperature.

Heat Treatment of Bacteria

Bacteria are killed by heat treatment in order to minimise the risk ofcontamination of the artificial diet in the test plates. However, heattreatment of bacteria expressing double-stranded RNA is not aprerequisite for inducing toxicity towards the insects due to RNAinterference. The induced bacterial culture is centrifuged at 3000 g atroom temperature for 10 minutes, the supernatant discarded and thepellet subjected to 80° C. for 20 minutes in a water bath. After heattreatment, the bacterial pellet is resuspended in 1.5 ml MilliQ waterand the suspension transferred to a microfuge tube. Several tubes areprepared and used in the bioassays for each refreshment. The tubes arestored at −20° C. until further use.

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LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090306189A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

1.-18. (canceled)
 19. An isolated polynucleotide sequence selected fromthe group consisting of a polynucleotide sequence comprising a nucleicacid sequence set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 49-158, 159, 160, 163, 168, 173, 178, 183, 188, 193, 198,203, 208, 215, 220, 225, 230, 247, 249, 251, 253, 255, 257, 259,275-472, 473, 478, 483, 488, 493, 498, 503, 513, 515, 517, 519, 521,533-575, 576, 581, 586, 591, 596, 601, 603, 605, 607, 609, 621-767, 768,773, 778, 783, 788, 793, 795, 797, 799, 801, 813-862, 863, 868, 873,878, 883, 888, 890, 892, 894, 896, 908-1040, 1041, 1046, 1051, 1056,1061, 1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091,1093, 1095, 1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113,1161-1571, 1572, 1577, 1582, 1587, 1592, 1597, 1602, 1607, 1612, 1617,1622, 1627, 1632, 1637, 1642, 1647, 1652, 1657, 1662, 1667, 1672, 1677,1682, 1684, 1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704,1730-2039, 2040, 2045, 2050, 2055, 2060, 2065, 2070, 2075, 2080, 2085,2090, 2095, 2100, 2102, 2104, 2106, 2108, 2120-2338, 2339, 2344, 2349,2354, 2359, 2364, 2366, 2368, 2370, 2372, 2384-2460, 2461, 2466, 2471,2476 and 2481; a polynucleotide sequence having at least 70% sequenceidentity to a nucleic acid sequence set forth in SEQ ID NOs: 1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 49-158, 159, 160, 163, 168, 173, 178,183, 188, 193, 198, 203, 208, 215, 220, 225, 230, 247, 249, 251, 253,255, 257, 259, 275-472, 473, 478, 483, 488, 493, 498, 503, 513, 515,517, 519, 521, 533-575, 576, 581, 586, 591, 596, 601, 603, 605, 607,609, 621-767, 768, 773, 778, 783, 788, 793, 795, 797, 799, 801, 813-862,863, 868, 873, 878, 883, 888, 890, 892, 894, 896, 908-1040, 1041, 1046,1051, 1056, 1061, 1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087,1089, 1091, 1093, 1095, 1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111,1113, 1161-1571, 1572, 1577, 1582, 1587, 1592, 1597, 1602, 1607, 1612,1617, 1622, 1627, 1632, 1637, 1642, 1647, 1652, 1657, 1662, 1667, 1672,1677, 1682, 1684, 1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702,1704, 1730-2039, 2040, 2045, 2050, 2055, 2060, 2065, 2070, 2075, 2080,2085, 2090, 2095, 2100, 2102, 2104, 2106, 2108, 2120-2338, 2339, 2344,2349, 2354, 2359, 2364, 2366, 2368, 2370, 2372, 2384-2460, 2461, 2466,2471, 2476 and 2481; and a double stranded ribonucleotide sequenceproduced from the expression of a polynucleotide sequence, whereincontact of said ribonucleotide sequence by a pest inhibits the growth ofsaid pest.
 20. The ribonucleotide sequence of claim 19, wherein contactof said sequence inhibits expression of a nucleotide sequencesubstantially complementary to said sequence.
 21. A cell transformedwith a polynucleotide of any of claim
 19. 22. The cell of claim 21,wherein said cell is a bacterial, yeast, or algal cell.
 23. A foodproduct comprising the cell of claim
 22. 24. The food product of claim23, wherein said food product is selected from the group consisting ofstored grains, pet food, and powdered chocolate.
 25. A compositioncomprising the polynucleotide of claim
 19. 26. The composition of claim25, wherein said composition is selected from the group consisting of aspray, powder, pellet, gel, capsule, food product, garment bag, andbook.
 27. A method for controlling pest infestation, comprising exposinga pest to a composition comprising a polynucleotide sequence thatinhibits a pest biological activity.
 28. The method of claim 27, whereinsaid polynucleotide sequence is set forth in any of SEQ ID NOs: 1, 3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 49-158, 159, 160, 163, 168, 173, 178,183, 188, 193, 198, 203, 208, 215, 220, 225, 230, 247, 249, 251, 253,255, 257, 259, 275-472, 473, 478, 483, 488, 493, 498, 503, 513, 515,517, 519, 521, 533-575, 576, 581, 586, 591, 596, 601, 603, 605, 607,609, 621-767, 768, 773, 778, 783, 788, 793, 795, 797, 799, 801, 813-862,863, 868, 873, 878, 883, 888, 890, 892, 894, 896, 908-1040, 1041, 1046,1051, 1056, 1061, 1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087,1089, 1091, 1093, 1095, 1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111,1113, 1161-1571, 1572, 1577, 1582, 1587, 1592, 1597, 1602, 1607, 1612,1617, 1622, 1627, 1632, 1637, 1642, 1647, 1652, 1657, 1662, 1667, 1672,1677, 1682, 1684, 1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702,1704, 1730-2039, 2040, 2045, 2050, 2055, 2060, 2065, 2070, 2075, 2080,2085, 2090, 2095, 2100, 2102, 2104, 2106, 2108, 2120-2338, 2339, 2344,2349, 2354, 2359, 2364, 2366, 2368, 2370, 2372, 2384-2460, 2461, 2466,2471, 2476 and
 2481. 29. A pesticide comprising a polynucleotide ofclaim
 19. 30. A method for protecting an object from pest infestation,comprising treating the surface of said object with a compositioncomprising a polynucleotide of claim
 19. 31. The method of claim 30,wherein said object is selected from the group consisting of wood, tree,book binding, cloth, and a food storage container.
 32. A method forpreventing or treating an insect infestation comprising administering acomposition according to claim
 25. 33. A method for preventing ortreating a nematode infestation comprising administering a compositionaccording to claim
 25. 34. A method for preventing or treating a fungalinfection comprising administering a composition according to claim 25.